Human receptor tyrosine kinase mertk

ABSTRACT

Reagents that regulate human receptor tyrosine kinase MerTK and reagents which bind to human receptor tyrosine kinase MerTK gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, gastrointestinal and liver disorders, cancer, metabolic disorders (particularly diabetes), neurological disorders, cardiovascular disorders (particularly coronary heart disease), hematological disorders, reproductive disorders, endocrine and hormonal disorders, respiratory disorders (particularly COPD), and genitourinary disorders.

This application incorporates by reference co-pending U.S. provisionalapplication Ser. No. 60/391,933 filed Jun. 28, 2002 and Ser. No.60/432,669 filed Dec. 12, 2002.

FIELD OF THE INVENTION

The invention relates to the regulation of human receptor tyrosinekinase MerTK.

BACKGROUND OF THE INVENTION

Transmembrane receptor tyrosine kinases (RTKs) comprise a large andevolutionarily conserved family of structurally related proteins capableof transducing extracellular signals to the cytoplasm. U.S. Pat. No.5,998,187. The latent oncogenic potential of these molecules and themolecular mechanisms by which they function in signalling pathways havebeen the subject of extensive study.

In addition, genetic and biochemical analyses of a variety ofdevelopmental mutants have led to recognition of the pivotal rolesplayed by RTK-mediated signalling pathways in the regulation of celldetermination, migration, and proliferation. Notable examples inDrosophila include the role of sevenless and its ligand, bride ofsevenless, in R7 photoreceptor determination (Kramer, H., Cagan, R. L. &Zipursky, S. L. (1991), Nature, 352, 207-212), and of DER/fib in earlymorphogenetic events during gastrulation (Schejter, E. D. & Shilo, B.-Z.(1989), Cell, 56, 1093-1104). Similarly, in the mouse, loss of functionmutations at the W/c-kit (Geissler, E. N., Rayn, M. A. & Housman, D. E.(1988), Cell, 55, 185-192; Chabot, B., Stephenson, D. A., Chapman, V.M., Besmer, P. & Bernstein, A. (1988), Nature, 335, 88-89) and Sl(Russell, E. S. (1979), Adv. Genet., 20, 357-459) loci have revealed theimportance of the Kit receptor and its ligand in melanogenesis,hematopoiesis, and gametogenesis (Dubreuil, P., Rottapel, R., Reith, A.D., Forrester, L. & Bernstein, A. (1990), Ann. N.Y. Acad. Sci., 599,58-65; Williams, D. E., Eisenman, J., Baird, A., Rauch, C., Ness, K V.,March, C. J., Park, L. S., Martin, U., Mochizuki, D. Y., Boswell, H. S.,Burgess, G. S., Cosman, D. & Lyman, S. D. (1990), Cell, 63, 167-174;Copeland, N. G., Gilbert, D. J., Cho, B. C., Donovan, P. J., Jenkins, N.A., Cosman, D. Anderson, D., Lyman, S. D. & Williams, D. E. (1990),Cell, 63, 175-183 and Flanagan, J. G. & Leder, P. (1990), Cell, 63,185-194) while a deletion in the gene encoding PDGFR-.alpha. has beencorrelated with the Patch mutation, which also causes a defect inmelanogenesis (Stephenson, D. A., Mercola, M., Anderson, E., Wang, C.,Stiles, C. D., Bowen-Pope, D. F. & Chapman, V. M. (1991), Proc. Natl.Acad. Sci., 88, 6-10). These observations, together with others(reviewed in Pawson, T. & Bernstein, A. (1991), Trends Gen., 6,350-356), have established the importance of receptor-ligandinteractions in the regulation of development.

Angiogenesis in both the embryo and adult requires the differentiation,proliferation, and migration of endothelial cells. Tissuetransplantation studies with quail/chick chimeras have established thatthe developmental cues for both endothelial cell differentiation andproper patterning of vessels are extracellular and not pre-programmedwithin the cell (Noden, D. M. (1988) Development, 103, 121-140). Severalpeptide hormones, such as bFGF, VEGF and PD-EGF, have been shown to haveboth mitogenic and chemotactic effects on cultured endothelial cells(see Tomasi, V., Manica, F. & Spisni, E. (1990), BioFactors, 2, 213-217;Klagsbrun, M. & D'Amore, P. (1991), Annu. Rev. Physiol., 53, 217-239,for reviews). However, many of these factors also show similar effectson other cell types, implying that receptors for these factors are alsoexpressed by such cells.

Studies have demonstrated that both tyrosine kinase activity andphosphotyrosine-containing proteins are increased in embryonic chickenheart relative to the adult (Maher, P. A. (1991). J. Cell Biol., 112,955-963), and that inhibitors of kinase activity impede inductiveprocesses during in vitro differentiation of cardiac explants derivedfrom chicken embryos (Runyan, R. B., Potts, J. D., Sharma, R. V.,Loeber, C. P., Chiang, J. J. & Bhalla, R. C. (1990), Cell Reg., 1,301-313).

Because of the importance of receptor tyrosine kinases, there is a needin the art to identify additional such kinases, which can be regulatedto provide therapeutic effects.

It is an object of the invention to provide reagents and methods ofregulating a human receptor tyrosine kinase MerTK. This and otherobjects of the invention are provided by one or more of the embodimentsdescribed below.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide from the groupconsisting of:

-   a) a polynucleotide encoding a Receptor tyrosine kinase MerTK    polypeptide comprising an amino acid sequence selected from the    group consisting of: amino acid sequences which are at least about    98% identical to the amino acid sequence shown in SEQ ID NO: 2; and

the amino acid sequence shown in SEQ ID NO: 2.

b) a polynucleotide comprising the sequence of SEQ ID NO: 1 or 3;

c) a polynucleotide which hybridizes under stringent conditions to apolynucleotide specified in (a) and (b) and encodes a Receptor tyrosinekinase MerTK polypeptide;

-   d) a polynucleotide the sequence of which deviates from the    polynucleotide sequences specified in (a) to (c) due to the    degeneration of the genetic code and encodes a Receptor tyrosine    kinase MerTK polypeptide; and-   e) a polynucleotide which represents a fragment, derivative or    allelic variation of a polynucleotide sequence specified in (a)    to (d) and encodes a Receptor tyrosine kinase MerTK polypeptide.

A novel human receptor tyrosine kinase MerTK is a discovery of thepresent invention. Human receptor tyrosine kinase MerTK comprises theamino acid sequence shown in SEQ ID NO:2. A DNA sequence harbouring thecoding sequence (ORF) for human receptor tyrosine kinase MerTK is shownin SEQ ID NO:1. This sequence is located on chromosome 2q13. The ORF isshown in SEQ ID NO:3. Related ESTs (SEQ ID NOs: 6-17) are expressed inpooled colon, kidney, and stomach cells, duodenal adenocarcinoma (cellline), osteosarcoma (cell line), teratocarcinoma, eye, anaplasticoligodendroglioma, normal placenta, trabecular meshwork, and two pooledhigh-grade transitional cell tumors.

The 3D structure clearly infers homology to proto-oncogenetyrosine-protein kinase abl fragment. A kinase domain, fibronectin typeIII domains, and immunoglobulin domains are shown. Furthermore, tyrosinekinase and ATP-binding regions are shown. The active site D residue andATP binding site K residue are conserved in the sequence.

Human receptor tyrosine kinase MerTK of the invention is expected to beuseful for the same purposes as previously identified receptor tyrosinekinase MerTK enzymes. Human receptor tyrosine kinase MerTK is believedto be useful in therapeutic methods to treat disorders such asgastrointestinal and liver disorders, cancer, metabolic disorders(particularly diabetes), neurological disorders, cardiovasculardisorders (particularly coronary heart disease), hematologicaldisorders, reproductive disorders, endocrine and hormonal disorders,respiratory disorders (particularly COPD), and genitourinary disorders.Human receptor tyrosine kinase MerTK also can be used to screen forhuman receptor tyrosine kinase MerTK activators and inhibitors.

One embodiment of the present invention is an expression vectorcontaining any polynucleotide of the present invention.

Yet another embodiment of the present invention is a host cellcontaining any expression vector of the present invention.

Still another embodiment of the present invention is a substantiallypurified Receptor tyrosine kinase MerTK polypeptide encoded by anypolynucleotide of the present invention.

Even another embodiment of the present invention is a method ofproducing a Receptor tyrosine kinase MerTK polypeptide of the presentinvention, wherein the method comprises the following steps:

-   a. culturing the host cells of the present invention under    conditions suitable for the expression of the Receptor tyrosine    kinase MerTK polypeptide; and-   b. recovering the Receptor tyrosine kinase MerTK polypeptide from    the host cell culture.

Yet another embodiment of the present invention is a method fordetecting a polynucleotide encoding a Receptor tyrosine kinase MerTKpolypeptide in a biological sample comprising the following steps:

-   a. hybridizing any polynucleotide of the present invention to a    nucleic acid material of a biological sample, thereby forming a    hybridization complex; and-   b. detecting said hybridization complex.

Still another embodiment of the present invention is a method fordetecting a polynucleotide of the present invention or a Receptortyrosine kinase MerTK polypeptide of the present invention comprisingthe steps of:

-   a. contacting a biological sample with a reagent which specifically    interacts with the polynucleotide or the Receptor tyrosine kinase    MerTK polypeptide and-   b. detecting the interaction

Even another embodiment of the present invention is a diagnostic kit forconducting any method of the present invention.

Yet another embodiment of the present invention is a method of screeningfor agents which decrease the activity of a Receptor tyrosine kinaseMerTK, comprising the steps of:

-   a. contacting a test compound with a Receptor tyrosine kinase MerTK    polypeptide encoded by any polynucleotide of the present invention;-   b. detecting binding of the test compound to the Receptor tyrosine    kinase MerTK polypeptide, wherein a test compound which binds to the    polypeptide is identified as a potential therapeutic agent for    decreasing the activity of a Receptor tyrosine kinase MerTK

Still another embodiment of the present invention is a method ofscreening for agents which regulate the activity of a Receptor tyrosinekinase MerTK, comprising the steps of:

-   a. contacting a test compound with a Receptor tyrosine kinase MerTK    polypeptide encoded by any polynucleotide of the present invention;    and-   b. detecting a Receptor tyrosine kinase MerTK activity of the    polypeptide, wherein a test compound which increases the Receptor    tyrosine kinase MerTK activity is identified as a potential    therapeutic agent for increasing the activity of the Receptor    tyrosine kinase MerTK, and wherein a test compound which decreases    the Receptor tyrosine kinase MerTK activity of the polypeptide is    identified as a potential therapeutic agent for decreasing the    activity of the Receptor tyrosine kinase MerTK.

Even another embodiment of the present invention is a method ofscreening for agents which decrease the activity of a Receptor tyrosinekinase MerTK, comprising the step of:

contacting a test compound with any polynucleotide of the presentinvention and detecting binding of the test compound to thepolynucleotide, wherein a test compound which binds to thepolynucleotide is identified as a potential therapeutic agent fordecreasing the activity of Receptor tyrosine kinase MerTK.

Yet another embodiment of the present invention is a method of reducingthe activity of a Receptor tyrosine kinase MerTK, comprising the stepof:

contacting a cell with a reagent which specifically binds to anypolynucleotide of the present invention or any Receptor tyrosine kinaseMerTK polypeptide of the present invention, whereby the activity ofReceptor tyrosine kinase MerTK is reduced.

Still another embodiment of the present invention is a reagent thatmodulates the activity of a Receptor tyrosine kinase MerTK polypeptideor a polynucleotide wherein said reagent is identified by any methods ofthe present invention.

Even another embodiment of the present invention is a pharmaceuticalcomposition, comprising:

an expression vector of the present invention or a reagent of thepresent invention and a pharmaceutically acceptable carrier.

Yet another embodiment of the present invention is the use of anexpression vector of the present invention or a reagent of the presentinvention for modulating the activity of a Receptor tyrosine kinaseMerTK in a disease, preferably a gastrointestinal and liver disorder,cancer, a metabolic disorder (particularly diabetes), a neurologicaldisorder, a cardiovascular disorder (particularly coronary heartdisease), a hematological disorder, a reproductive disorder, anendocrine and hormonal disorder, a respiratory disorders (particularlyCOPD) or a genitourinary disorder.

The invention thus provides a human receptor tyrosine kinase MerTK thatcan be used to identify test compounds that may act, for example, asactivators or inhibitors at the enzyme's active site. Human receptortyrosine kinase MerTK and fragments thereof also are useful in raisingspecific antibodies that can block the enzyme and effectively reduce itsactivity.

Polypeptides

Human receptor tyrosine kinase MerTK polypeptides according to theinvention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, or1016 contiguous amino acids selected from the amino acid sequence shownin SEQ ID NO:2 or a biologically active variant thereof, as definedbelow. A receptor tyrosine kinase MerTK polypeptide of the inventiontherefore can be a portion of a receptor tyrosine kinase MerTK protein,a full-length receptor tyrosine kinase MerTK protein, or a fusionprotein comprising all or a portion of a receptor tyrosine kinase MerTKprotein.

Biologically Active Variants

Human receptor tyrosine kinase MerTK polypeptide variants which arebiologically active, e.g., retain a receptor tyrosine kinase activity,also are human receptor tyrosine kinase MerTK polypeptides. Preferably,naturally or non-naturally occurring human receptor tyrosine kinaseMerTK polypeptide variants have amino acid sequences which are at leastabout 98 or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof. Percent identity between a putative humanreceptor tyrosine kinase MerTK polypeptide variant and an amino acidsequence of SEQ ID NO:2 is determined by conventional methods. See, forexample, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff &Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two aminoacid sequences are aligned to optimize the alignment scores using a gapopening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62”scoring matrix of Henikoff & Henikoff, 1992.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “TASTA”similarity search algorithm of Pearson & Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant. The FASTA algorithm is described by Pearson & Lipman, Proc.Nat'l Acad. Sci. USA 85:2444(1988), and by Pearson, Meth. Enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO: 2)and a test sequence that have either the highest density of identities(if the ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence the ktup value), then the trimmed initial regionsare examined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman & Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variations in percent identity can be due, for example, to amino acidsubstitutions, insertions, or deletions. Amino acid substitutions aredefined as one for one amino acid replacements. They are conservative innature when the substituted amino acid has similar structural and/orchemical properties. Examples of conservative replacements aresubstitution of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of a human receptor tyrosine kinase MerTKpolypeptide can be found using computer programs well known in the art,such as DNASTAR software.

The invention additionally, encompasses receptor tyrosine kinase MerTKpoly-peptides that are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications can be carried outby known techniques including, but not limited, to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The receptor tyrosine kinase MerTK polypeptides may also bemodified with a detectable label, such as an enzymatic, fluorescent,isotopic or affinity label to allow for detection and isolation of theprotein.

The invention also provides chemically modified derivatives of receptortyrosine kinase MerTK polypeptides that may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization can be selectedfrom water soluble polymers-such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol, and the like. The polypeptides can be modified atrandom or predetermined positions within the molecule and can includeone, two, three, or more attached chemical moieties.

Whether an amino acid change or a polypeptide modification results in abiologically active receptor tyrosine kinase MerTK polypeptide canreadily be determined by assaying for enzymatic activity, as describedfor example, in Example 4.

Fusion Proteins

Fusion proteins are useful for generating antibodies against receptortyrosine kinase MerTK polypeptide amino acid sequences and for use invarious assay systems. For example, fusion proteins can be used toidentify proteins that interact with portions of a human receptortyrosine kinase MerTK polypeptide. Protein affinity chromatography orlibrary-based assays for protein-protein interactions, such as the yeasttwo-hybrid or phage display systems, can be used for this purpose. Suchmethods are well known in the art and also can be used as drug screens.

A human receptor tyrosine kinase MerTK polypeptide fusion proteincomprises two polypeptide segments fused together by means of a peptidebond. The first polypeptide segment comprises a receptor tyrosine kinaseMerTK polypeptide, such as those described above. The first polypeptidesegment also can comprise full-length receptor tyrosine kinase MerTKprotein.

The second polypeptide segment can be a full-length protein or a proteinfragment. Proteins commonly used in fusion protein construction includeβ-galactosidase, βglucuronidase, green fluorescent protein (GFP),autofluorescent proteins, including blue fluorescent protein (BFP),glutathione-S-transferase (GST), luciferase, horse-radish peroxidase(HRP), and chloramphenicol acetyltransferase (CAT). Additionally,epitope tags are used in fusion protein constructions, includinghistidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructionscan include maltose binding protein (MBP), S-tag, Lex a DNA bindingdomain (DBD) fusions, GAL4 DNA binding domain fusions, and herpessimplex virus (HSV) BP16 protein fusions. A fusion protein also can beengineered to contain a cleavage site located between the receptortyrosine kinase MerTK polypeptide-encoding sequence and the heterologousprotein sequence, so that the receptor tyrosine kinase MerTK polypeptidecan be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art.Preferably, a fusion protein is produced by covalently linking twopolypeptide segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be used to prepare fusion proteins,for example, by making a DNA construct which comprises coding sequencesselected from SEQ ID NO:1 in proper reading frame with nucleotidesencoding the second polypeptide segment and expressing the DNA constructin a host cell, as is known in the art. Many kits for constructingfusion proteins are available from companies such as Promega Corporation(Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View,Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBLInternational Corporation (MIC; Watertown, Mass.), and QuantumBiotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of human receptor tyrosine kinase MerTK polypeptide canbe obtained using receptor tyrosine kinase MerTK polypeptidepolynucleotides (described below) to make suitable probes or primers forscreening cDNA expression libraries from other species, such as mice,monkeys, or yeast, identifying cDNAs which encode homologs of receptortyrosine kinase MerTK polypeptide, and expressing the cDNAs as is knownin the art.

Polynucleotides

A human receptor tyrosine kinase MerTK polynucleotide can be single- ordouble-stranded and comprises a coding sequence or the complement of acoding sequence for a receptor tyrosine kinase MerTK polypeptide. Acoding sequence for human receptor tyrosine kinase MerTK is shown in SEQID NO:3.

Degenerate nucleotide sequences encoding human receptor tyrosine kinaseMerTK polypeptides, as well as homologous nucleotide sequences which areat least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or99% identical to the nucleotide sequence shown in SEQ ID NO:1 or 3 ortheir complements also are receptor tyrosine kinase MerTKpolynucleotides. Percent sequence identity between the sequences of twopolynucleotides is determined using computer programs such as ALIGNwhich employ the FASTA algorithm, using an affine gap search with a gapopen penalty of −12 and a gap extension penalty of −2. Complementary DNA(cDNA) molecules, species homologs, and variants of receptor tyrosinekinase MerTK polynucleotides that encode biologically active receptortyrosine kinase MerTK polypeptides also are receptor tyrosine kinaseMerTK polynucleotides. Polynucleotide fragments comprising at least 8,9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:1 or 3or their complements also are receptor tyrosine kinase MerTKpolynucleotides. These fragments can be used, for example, ashybridization probes or as antisense oligonucleotides.

Identification of Polynucleotide Variants and Homologs

Variants and homologs of the receptor tyrosine kinase MerTKpolynucleotides described above also are receptor tyrosine kinase MerTKpolynucleotides. Typically, homologous receptor tyrosine kinase MerTKpolynucleotide sequences can be identified by hybridization of candidatepolynucleotides to known receptor tyrosine kinase MerTK polynucleotidesunder stringent conditions, as is known in the art. For example, usingthe following wash conditions—2×SSC (0.3 M NaCl, 0.03 M sodium citrate,pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC,0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice,10 minutes each—homologous sequences can be identified which contain atmost about 25-30% basepair mismatches. More preferably, homologousnucleic acid strands contain 15-25% basepair mismatches, even morepreferably 5-15% basepair mismatches.

Species homologs of the receptor tyrosine kinase MerTK polynucleotidesdisclosed herein also can be identified by making suitable probes orprimers and screening cDNA expression libraries from other species, suchas mice, monkeys, or yeast. Human variants of receptor tyrosine kinaseMerTK polynucleotides can be identified, for example, by screening humancDNA expression libraries. It is well known that the T_(m) of adouble-stranded DNA decreases by 1-1.5° C. with every 1% decrease inhomology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of humanreceptor tyrosine kinase MerTK polynucleotides or receptor tyrosinekinase MerTK polynucleotides of other species can therefore beidentified by hybridizing a putative homologous receptor tyrosine kinaseMerTK polynucleotide with a polynucleotide having a nucleotide sequenceof SEQ ID NO:1 or 3 or the complement thereof to form a test hybrid. Themelting temperature of the test hybrid is compared with the meltingtemperature of a hybrid comprising polynucleotides having perfectlycomplementary nucleotide sequences, and the number or percent ofbasepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to receptor tyrosine kinase MerTKpoly-nucleotides or their complements following stringent hybridizationand/or wash conditions also are receptor tyrosine kinase MerTKpolynucleotides. Stringent wash conditions are well known and understoodin the art and are disclosed, for example, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination oftemperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a receptor tyrosine kinase MerTKpolynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or 3 orthe complement thereof and a polynucleotide sequence which is at leastabout 50, preferably about 75, 90, 96, or 98% identical to one of thosenucleotide sequences can be calculated, for example, using the equationof Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(%G+C)−0.63(% formamide)−600/l),where l=the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4×SSC at 65° C., or 50%formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

Preparation of Polynucleotides

A human receptor tyrosine kinase MerTK polynucleotide can be isolatedfree of other cellular components such as membrane components, proteins,and lipids. Poly-nucleotides can be made by a cell and isolated usingstandard nucleic acid purification techniques, or synthesized using anamplification technique, such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide can be used to obtain isolated receptor tyrosine kinaseMerTK polynucleotides. For example, restriction enzymes and probes canbe used to isolate polynucleotide fragments, which comprise receptortyrosine kinase MerTK nucleotide sequences. Isolated polynucleotides arein preparations that are free or at least 70, 80, or 90% free of othermolecules.

Human receptor tyrosine kinase MerTK cDNA molecules can be made withstandard molecular biology techniques, using receptor tyrosine kinaseMerTK mRNA as a template. Human receptor tyrosine kinase MerTK cDNAmolecules can thereafter be replicated using molecular biologytechniques known in the art and disclosed in manuals such as Sambrook etal. (1989). An amplification technique, such as PCR, can be used toobtain additional copies of polynucleotides of the invention, usingeither human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizereceptor tyrosine kinase MerTK polynucleotides. The degeneracy of thegenetic code allows alternate nucleotide sequences to be synthesizedwhich will encode a human receptor tyrosine kinase MerTK polypeptidehaving, for example, an amino acid sequence shown in SEQ ID NO:2 or abiologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend the nucleic acidsequences disclosed herein to detect upstream sequences such aspromoters and regulatory elements. For example, restriction-site PCRuses universal primers to retrieve unknown sequence adjacent to a knownlocus. Sarkar, PCR Methods Applic. 2, 318-322, 1993; Triglia et al.,Nucleic Acids Res. 16, 8186, 1988; Lagerstrom et al., PCR MethodsApplic. 1, 111-119, 1991; Parker et al., Nucleic Acids Res. 19,3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDERlibraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA(CLONTECH, Palo Alto, Calif.). See WO 01/98340

Obtaining Polypeptides

Human receptor tyrosine kinase MerTK polypeptides can be obtained, forexample, by purification from human cells, by expression of receptortyrosine kinase MerTK polynucleotides, or by direct chemical synthesis.

Protein Purification

Human receptor tyrosine kinase MerTK polypeptides can be purified fromany human cell that expresses the receptor, including host cells whichhave been transfected with receptor tyrosine kinase MerTKpolynucleotides. A purified receptor tyrosine kinase MerTK polypeptideis separated from other compounds that normally associate with thereceptor tyrosine kinase MerTK polypeptide in the cell, such as certainproteins, carbohydrates, or lipids, using methods well-known in the art.Such methods include, but are not limited to, size exclusionchromatography, ammonium sulfate fractionation, ion exchangechromatography, affinity chromatography, and preparative gelelectrophoresis.

A preparation of purified receptor tyrosine kinase MerTK polypeptides isat least 80% pure; preferably, the preparations are 90%, 95%, or 99%pure. Purity of the preparations can be assessed by any means known inthe art, such as SDS-polyacrylamide gel electrophoresis.

Expression of Polynucleotides

To express a human receptor tyrosine kinase MerTK polynucleotide, thepolynucleotide can be inserted into an expression vector which containsthe necessary elements for the transcription and translation of theinserted coding sequence. Methods which are well known to those skilledin the art can be used to construct expression vectors containingsequences encoding receptor tyrosine kinase MerTK polypeptides andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed, for example, in Sambrook et al. (1989) and in Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., 1989.

A variety of expression vector/host systems can be utilized to containand express sequences encoding a human receptor tyrosine kinase MerTKpolypeptide. These include, but are not limited to, microorganisms, suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors, insect cell systems infected with virus expression vectors(e.g., baculovirus), plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322plasmids), or animal cell systems. See WO 01/98340.

Host Cells

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedreceptor tyrosine kinase MerTK polypeptide in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells that havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38)are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.See WO 01/98340.

Alternatively, host cells which contain a human receptor tyrosine kinaseMerTK polynucleotide and which express a human receptor tyrosine kinaseMerTK polypeptide can be identified by a variety of procedures known tothose of skill in the art. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmuno-assay (RIA), and fluorescence activated cellsorting (FACS). Hampton et al., SEROLOGICAL METHODS: A LABORATORYMANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med158, 1211-1216, 1983). See WO 01/98340.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding receptortyrosine kinase MerTK polypeptides include oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, sequences encoding a human receptor tyrosinekinase MerTK polypeptide can be cloned into a vector for the productionof an “mRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Pharmacia Biotech, Promega, and USBiochemical). Suitable reporter molecules or labels which can be usedfor ease of detection include radionuclides, enzymes, and fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a humanreceptor tyrosine kinase MerTK polypeptide can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The polypeptide produced by a transformed cell can besecreted or contained intracellularly depending on the sequence and/orthe vector used. As will be understood by those of skill in the art,expression vectors containing polynucleotides which encode receptortyrosine kinase MerTK polypeptides can be designed to contain signalsequences which direct secretion of soluble receptor tyrosine kinaseMerTK polypeptides through a prokaryotic or eukaryotic cell membrane orwhich direct the membrane insertion of membrane-bound receptor tyrosinekinase MerTK polypeptide. See WO 01/98340.

Chemical Synthesis

Sequences encoding a human receptor tyrosine kinase MerTK polypeptidecan be synthesized, in whole or in part, using chemical methods wellknown in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser.215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).Alternatively, a human receptor tyrosine kinase MerTK polypeptide itselfcan be produced using chemical methods to synthesize its amino acidsequence, such as by direct peptide synthesis using solid-phasetechniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Robergeet al., Science 269, 202-204, 1995). Protein synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Optionally, fragments of receptor tyrosine kinase MerTKpolypeptides can be separately synthesized and combined using chemicalmethods to produce a full-length molecule. See WO 01/98340.

As will be understood by those of skill in the art, it may beadvantageous to produce receptor tyrosine kinase MerTKpolypeptide-encoding nucleotide sequences possessing non-naturallyoccurring codons. For example, codons preferred by a particularprokaryotic or eukaryotic host can be selected to increase the rate ofprotein expression or to produce an RNA transcript having desirableproperties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter receptor tyrosine kinaseMerTK polypeptide-encoding sequences for a variety of reasons, includingbut not limited to, alterations which modify the cloning, processing,and/or expression of the polypeptide or mRNA product. DNA shuffling byrandom fragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides can be used to engineer the nucleotide sequences. Forexample, site-directed mutagenesis can be used to insert new restrictionsites, alter glycosylation patterns, change codon preference, producesplice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bindspecifically to an epitope of a human receptor tyrosine kinase MerTKpolypeptide. “Antibody” as used herein includes intact immunoglobulinmolecules, as well as fragments thereof, such as Fab, F(ab′)₂, and Fv,which are capable of binding an epitope of a human receptor tyrosinekinase MerTK polypeptide. Typically, at least 6, 8, 10, or 12 contiguousamino acids are required to form an epitope. However, epitopes whichinvolve non-contiguous amino acids may require more, e.g., at least 15,25, or 50 amino acids.

An antibody which specifically binds to an epitope of a human receptortyrosine kinase MerTK polypeptide can be used therapeutically, as wellas in immuno-chemical assays, such as Western blots, ELISAs,radioimmunoassays, immunohistochemical assays, immunoprecipitations, orother immunochemical assays known in the art. Various immunoassays canbe used to identify antibodies having the desired specificity. Numerousprotocols for competitive binding or immunoradiometric assays are wellknown in the art. Such immunoassays typically involve the measurement ofcomplex formation between an immunogen and an antibody that specificallybinds to the immunogen.

Typically, an antibody that specifically binds to a human receptortyrosine kinase MerTK polypeptide provides a detection signal at least5-, 10-, or 20-fold higher than a detection signal provided with otherproteins when used in an immunochemical assay. Preferably, antibodiesthat specifically bind to receptor tyrosine kinase MerTK polypeptides donot detect other proteins in immunochemical assays and canimmunoprecipitate a human receptor tyrosine kinase MerTK polypeptidefrom solution. See WO 01/98340.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences that arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level ofreceptor tyrosine kinase MerTK gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides,or a combination of both. Oligonucleotides can be synthesized manuallyor by an automated synthesizer, by covalently linking the 5′ end of onenucleotide with the 3′ end of another nucleotide with non-phosphodiesterinternucleotide linkages such alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, alkylphosphonates,phosphoramidates, phosphate esters, carbamates, acetaniidate,carboxymethyl esters, carbonates, and phosphate triesters. See Brown,Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72,1994; Uhlnann et al., Chem. Rev. 90, 543-583, 1990.

Modifications of receptor tyrosine kinase MerTK gene expression can beobtained by designing antisense oligonucleotides that will form duplexesto the control, 5′, or regulatory regions of the receptor tyrosinekinase MerTK gene. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orchaperons. Therapeutic advances using triplex DNA have been described inthe literature (e.g., Gee et al., in Huber & Carr, MOLECULAR ANDIMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994).An antisense oligonucleotide also can be designed to block translationof mRNA by preventing the transcript from binding to ribosomes. See WO01/98340.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech,Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568;1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture &Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used toinhibit gene function by cleaving an RNA sequence, as is known in theart (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Examples include engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of specific nucleotide sequences.

The coding sequence of a human receptor tyrosine kinase MerTKpolynucleotide can be used to generate ribozymes that will specificallybind to mRNA transcribed from the receptor tyrosine kinase MerTKpolynucleotide. Methods of designing and constructing ribozymes whichcan cleave other RNA molecules in trans in a highly sequence specificmanner have been developed and described in the art (see Haseloff et al.Nature 334, 585-591, 1988). For example, the cleavage activity ofribozymes can be targeted to specific RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target RNA and thusspecifically hybridizes with the target (see, for example, Gerlach etal.” EP 321,201). See WO 01/98340.

Differentially Expressed Genes

Described herein are methods for the identification of genes whoseproducts interact with human receptor tyrosine kinase MerTK. Such genesmay represent genes that are differentially expressed in disordersincluding, but not limited to, gastrointestinal and liver disorders,cancer, metabolic disorders (particularly diabetes), neurologicaldisorders, cardiovascular disorders (particularly coronary heartdisease), hematological disorders, reproductive disorders, endocrine andhormonal disorders, respiratory disorders (particularly COPD), andgenitourinary disorders. Further, such genes may represent genes thatare differentially regulated in response to manipulations relevant tothe progression or treatment of such diseases. Additionally, such genesmay have a temporally modulated expression, increased or decreased atdifferent stages of tissue or organism development. A differentiallyexpressed gene may also have its expression modulated under controlversus experimental conditions. In addition, the human receptor tyrosinekinase MerTK gene or gene product may itself be tested for differentialexpression.

The degree to which expression differs in a normal versus a diseasedstate need only be large enough to be visualized via standardcharacterization techniques such as differential display techniques.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase), PCR, and Northern analysis.

To identify differentially expressed genes total RNA or, preferably,mRNA is isolated from tissues of interest. For example, RNA samples areobtained from tissues of experimental subjects and from correspondingtissues of control subjects. Any RNA isolation technique that does notselect against the isolation of mRNA may be utilized for thepurification of such RNA samples. See, for example, Ausubel et al., ed.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. NewYork, 1987-1993. Large numbers of tissue samples may readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,U.S. Pat. No. 4,843,155.

Transcripts within the collected RNA samples that represent RNA producedby differentially expressed genes are identified by methods well knownto those of skill in the art. They include, for example, differentialscreening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85, 208-12,1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53;Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and,preferably, differential display (Liang & Pardee, Science 257, 967-71,1992; U.S. Pat. No. 5,262,311).

The differential expression information may itself suggest relevantmethods for the treatment of disorders involving the human receptortyrosine kinase MerTK. For example, treatment may include a modulationof expression of the differentially expressed genes and/or the geneencoding the human receptor tyrosine kinase MerTK. The differentialexpression information may indicate whether the expression or activityof the differentially expressed gene or gene product or the humanreceptor tyrosine kinase MerfK gene or gene product are up-regulated ordown-regulated.

Screening Methods

The invention provides assays for screening test compounds that bind toor modulate the activity of a human receptor tyrosine kinase MerTKpolypeptide or a human receptor tyrosine kinase MerTK polynucleotide. Atest compound preferably binds to a human receptor tyrosine kinase MerTKpolypeptide or polynucleotide. More preferably, a test compounddecreases or increases enzymatic activity by at least about 10,preferably about 50, more preferably about 75, 90, or 100% relative tothe absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art orcan be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, test compounds can be obtainedusing any of the numerous combinatorial library methods known in theart, including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in theart (see, for example, DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90,6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994;Zuckermann et al., J. Med Chem 37, 2678, 1994; Cho et al., Science 261,1303, 1993; Carell et al., Angew. Chem. Int. Ed Engl. 33, 2059, 1994;Carell et al., Angew. Chem. Int. Ed Engl. 33, 2061; Gallop et al., J.Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented insolution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or onbeads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556,1993), bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids(Cull et al., Proc. Natl. Acad. Sci. USA. 89, 1865-1869, 1992), or phage(Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249,404-406, 1990); Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-6382,1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Pat.No. 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to receptortyrosine kinase MerTK polypeptides or polynucleotides or to affectreceptor tyrosine kinase MerTK activity or receptor tyrosine kinaseMerTK gene expression using high throughput screening. Using highthroughput screening, many discrete compounds can be tested in parallelso that large numbers of test compounds can be quickly screened. Themost widely established techniques utilize 96-well microtiter plates.The wells of the microtiter plates typically require assay volumes thatrange from 50 to 500 μl. In addition to the plates, many instruments,materials, pipettors, robotics, plate washers, and plate readers arecommercially available to fit the 96-well format.

Alternatively, “free format assays,” or assays that have no physicalbarrier between samples, can be used. For example, an assay usingpigment cells (melanocytes) in a simple homogeneous assay forcombinatorial peptide libraries is described by Jayawickreme et al.,Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placedunder agarose in petri dishes, then beads that carry combinatorialcompounds are placed on the surface of the agarose. The combinatorialcompounds are partially released the compounds from the beads. Activecompounds can be visualized as dark pigment areas because, as thecompounds diffuse locally into the gel matrix, the active compoundscause the cells to change colors.

Another example of a free format assay is described by Chelsky,“Strategies for Screening Combinatorial Libraries: Novel and TraditionalApproaches,” reported at the First Annual Conference of The Society forBiomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelskyplaced a simple homogenous enzyme assay for carbonic anhydrase inside anagarose gel such that the enzyme in the gel would cause a color changethroughout the gel. Thereafter, beads carrying combinatorial compoundsvia a photolinker were placed inside the gel and the compounds werepartially released by UV-light. Compounds that inhibited the enzyme wereobserved as local zones of inhibition having less color change.

Yet another example is described by Salmon et al., Molecular Diversity2, 57-63 (1996). In this example, combinatorial libraries were screenedfor compounds that had cytotoxic effects on cancer cells growing inagar.

Another high throughput screening method is described in Beutel et al.,U.S. Pat. No. 5,976,813. In this method, test samples are placed in aporous matrix. One or more assay components are then placed within, ontop of, or at the bottom of a matrix such as a gel, a plastic sheet, afilter, or other form of easily manipulated solid support. When samplesare introduced to the porous matrix they diffuse sufficiently slowly,such that the assays can be performed without the test samples runningtogether.

Binding Assays

For binding assays, the test compound is preferably a small moleculethat binds to and occupies, for example, the active site of the receptortyrosine kinase MerTK polypeptide, such that normal biological activityis prevented. Examples of such small molecules include, but are notlimited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the receptor tyrosinekinase MerTK polypeptide can comprise a detectable label, such as afluorescent, radioisotopic, chemiluminescent, or enzymatic label, suchas horseradish peroxidase, alkaline phosphatase, or luciferase.Detection of a test compound that is bound to the receptor tyrosinekinase MerTK polypeptide can then be accomplished, for example, bydirect counting of radioemmission, by scintillation counting, or bydetermining conversion of an appropriate substrate to a detectableproduct.

Alternatively, binding of a test compound to a human receptor tyrosinekinase MerTK polypeptide can be determined without labeling either ofthe interactants. For example, a microphysiometer can be used to detectbinding of a test compound with a human receptor tyrosine kinase MerTKpolypeptide. A microphysiometer (e.g., Cytosensor™) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a test compound and a human receptor tyrosine kinaseMerTK polypeptide (McConnell et al., Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a human receptortyrosine kinase MerTK polypeptide also can be accomplished using atechnology such as real-time Bimolecular Interaction Analysis (BIA)(Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo etal., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technologyfor studying biospecific interactions in real time, without labeling anyof the interactants (e.g., BLAcore™). Changes in the optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In yet another aspect of the invention, a human receptor tyrosine kinaseMerTK polypeptide can be used as a “bait protein” in a two-hybrid assayor three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos etal., Cell 72, 223-232, 1993; Madura et al., J. Biol. Chem. 268,12046-12054, 1993; Bartel et al., BioTechniques 14, 920-924, 1993;Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and Brent WO94/10300), toidentify other proteins which bind to or interact with the receptortyrosine kinase MerTK polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding ahuman receptor tyrosine kinase MerTK polypeptide can be fused to apolynucleotide encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct a DNA sequence that encodesan unidentified protein (“prey” or “sample”) can be fused to apolynucleotide that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract in vivo to form an protein-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ), which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected, and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the DNA sequenceencoding the protein that interacts with the receptor tyrosine kinaseMerTK polypeptide.

It may be desirable to immobilize either the receptor tyrosine kinaseMerTK polypeptide (or polynucleotide) or the test compound to facilitateseparation of bound from unbound forms of one or both of theinteractants, as well as to accommodate automation of the assay. Thus,either the receptor tyrosine kinase MerTK polypeptide (orpolynucleotide) or the test compound can be bound to a solid support.Suitable solid supports include, but are not limited to, glass orplastic slides, tissue culture plates, microtiter wells, tubes, siliconchips, or particles such as beads (including, but not limited to, latex,polystyrene, or glass beads). Any method known in the art can be used toattach the polypeptide (or polynucleotide) or test compound to a solidsupport, including use of covalent and non-covalent linkages, passiveabsorption, or pairs of binding moieties attached respectively to thepolypeptide (or polynucleotide) or test compound and the solid support.Test compounds are preferably bound to the solid support in an array, sothat the location of individual test compounds can be tracked. Bindingof a test compound to a human receptor tyrosine kinase MerTK polypeptide(or polynucleotide) can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and microcentrifuge tubes.

In one embodiment, the receptor tyrosine kinase MerTK polypeptide is afusion protein comprising a domain that allows the receptor tyrosinekinase MerTK polypeptide to be bound to a solid support. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbed receptortyrosine kinase MerTK polypeptide; the mixture is then incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components.Binding of the interactants can be determined either directly orindirectly, as, described above. Alternatively, the complexes can bedissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solidsupport also can be used in the screening assays of the invention. Forexample, either a human receptor tyrosine kinase MerTK polypeptide (orpolynucleotide) or a test compound can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated receptor tyrosinekinase MerTK polypeptides (or polynucleotides) or test compounds can beprepared from biotin-NHS(N-hydroxysuccinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.) and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical). Alternatively, antibodies which specifically bind toa receptor tyrosine kinase MerTK polypeptide, polynucleotide, or a testcompound, but which do not interfere with a desired binding site, suchas the active site of the receptor tyrosine kinase MerTK polypeptide,can be derivatized to the wells of the plate. Unbound target or proteincan be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies which specifically bind to the receptortyrosine kinase MerTK polypeptide or test compound, enzyme-linked assayswhich rely on detecting an activity of the receptor tyrosine kinaseMerTK polypeptide, and SDS gel electrophoresis under non-reducingconditions.

Screening for test compounds which bind to a human receptor tyrosinekinase MerTK polypeptide or polynucleotide also can be carried out in anintact cell. Any cell which comprises a receptor tyrosine kinase MerTKpolypeptide or polynucleotide can be used in a cell-based assay system.A receptor tyrosine kinase MerTK polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Binding of the test compound to a receptortyrosine kinase MerTK polypeptide or polynucleotide is determined asdescribed above.

Enzymatic Activity

Test compounds can be tested for the ability to increase or decrease theenzymatic activity of a human receptor tyrosine kinase MerTKpolypeptide. Enzymatic activity can be measured, for example, asdescribed in Example 4.

Enzyme assays can be carried out after contacting either a purifiedreceptor tyrosine kinase MerTK polypeptide, a cell membrane preparation,or an intact cell with a test compound. A test compound that decreasesenzymatic activity of a human receptor tyrosine kinase MerTK polypeptideby at least about 10, preferably about 50, more preferably about 75, 90,or 100% is identified as a potential therapeutic agent for decreasingreceptor tyrosine kinase MerTK activity. A test compound which increasesenzymatic activity of a human receptor tyrosine kinase MerTK polypeptideby at least about 10, preferably about 50, more preferably about 75, 90,or 100% is identified as a potential therapeutic agent for increasinghuman receptor tyrosine kinase MerTK activity.

Gene Expression

In another embodiment, test compounds that increase or decrease receptortyrosine kinase MerTK gene expression are identified. A receptortyrosine kinase MerTK polynucleotide is contacted with a test compound,and the expression of an RNA or polypeptide product of the receptortyrosine kinase MerTK polynucleotide is determined. The level ofexpression of appropriate mRNA or polypeptide in the presence of thetest compound is compared to the level of expression of mRNA orpolypeptide in the absence of the test compound. The test compound canthen be identified as a modulator of expression based on thiscomparison. For example, when expression of mRNA or polypeptide isgreater in the presence of the test compound than in its absence, thetest compound is identified as a stimulator or enhancer of the mRNA orpolypeptide expression. Alternatively, when expression of the mRNA orpolypeptide is less in the presence of the test compound than in itsabsence, the test compound is identified as an inhibitor of the mRNA orpolypeptide expression.

The level of receptor tyrosine kinase MerTK mRNA or polypeptideexpression in the cells can be determined by methods well known in theart for detecting mRNA or polypeptide. Either qualitative orquantitative methods can be used. The presence of polypeptide productsof a human receptor tyrosine kinase MerTK polynucleotide can bedetermined, for example, using a variety of techniques known in the art,including immunochemical methods such as radioimmunoassay, Westernblotting, and immunohistochemistry. Alternatively, polypeptide synthesiscan be determined in vivo, in a cell culture, or in an in vitrotranslation system by detecting incorporation of labeled amino acidsinto a human receptor tyrosine kinase MerTK polypeptide.

Such screening can be carried out either in a cell-free assay system orin an intact cell. Any cell that expresses a human receptor tyrosinekinase MerTK polynucleotide can be used in a cell-based assay system.The receptor tyrosine kinase MerTK polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Either a primary culture or an established cellline, such as CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions that can beadministered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise, for example,a human receptor tyrosine kinase MerTK polypeptide, receptor tyrosinekinase MerTK polynucleotide, ribozymes or antisense oligonucleotides,antibodies which specifically bind to a receptor tyrosine kinase MerTKpolypeptide, or mimetics, activators, or inhibitors of a human receptortyrosine kinase MerTK polypeptide activity. The compositions can beadministered alone or in combination with at least one other agent, suchas stabilizing compound, which can be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions can beadministered to a patient alone, or in combination with other agents,drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositionscan contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxy-propylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores can be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which also can contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments can be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions cancontain substances that increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds can be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers also can be used for delivery. Optionally, the suspensionalso can contain suitable stabilizers or agents that increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. For topical or nasal administration, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can befound in the latest edition of REMINGTON'S PHARMACEUICAL SCIENCES (MaackPublishing Co., Easton, Pa.). After pharmaceutical compositions havebeen prepared, they can be placed in an appropriate container andlabeled for treatment of an indicated condition. Such labeling wouldinclude amount, frequency, and method of administration.

Therapeutic Indications and Methods

Human receptor tyrosine kinase MerTK (MERTK) can be regulated to treatgastrointestinal and liver disorders, cancer, metabolic disordersparticularly diabetes), neurological disorders, cardiovascular disorders(particularly coronary heart disease), hematological disorders,reproductive disorders, endocrine and hormonal disorders, respiratorydisorders particularly COPD), and genitourinary disorders.

Hematological Disorders

Human MERTK is highly expressed in the following tissues of thehematological system: leukocytes (peripheral blood), bone marrow stromalcells, cord blood CD34⁺ cells, neutrophils cord blood, spleen, spleenliver cirrhosis. The expression in the above mentioned tissues and inparticular the differential expression between diseased tissue andhealthy tissue demonstrates that human MERTK protein or mRNA can be usedto diagnose hematological diseases. In addition, the activity of humanMERTK can be modulated to treat hematological disorders.

Anemia

Hemoglobin in red blood cells is the key component for transportingoxygen from the lungs to the tissues. In anemia the level of hemoglobinhas fallen below 12 g/L. Therefore the oxygen carrying capacity of bloodis reduced. Common reasons for anemia include acute or chronic bloodloss, insufficient levels of erythropoietin synthesis in the kidneys(e.g. in dialysis patients) or insufficient output of red blood cellsfrom bone marrow after chemotherapy or HIV infection etc. Currenttherapy of anemia is aimed at increasing the hematocrit either bytransfusion or by stimulating erythropoiesis with agents such aserythropoietin. The treatment goal is to restore hemoglobin level'sabove 12 g/L.

Neutropenia

Neutrophils play a key role in the defense against infections.Neutropenia is an abnormally low white blood cell count, which causes anincreased incidence of infections. Causes of neutropenia include:drug-induced (e.g., following cancer chemotherapy), increaseddestruction of neutrophils (e.g., immune-mediated) or decreased bonemarrow function (e.g., familial neutropenia). Neutropenia followingcancer chemotherapy is currently treated with growth factors such asG-CSF or GM-CSF that stimulate granulopoiesis. The treatment goal is toraise the neutrophil count in order to reduce the susceptibility toinfection.

Thrombocytopenia

Thrombocytopenia is a disorder where the number of platelets isinappropriately low. Since platelets play an essential role in thrombusformation to limit blood loss following vessel injury, insufficientplatelet levels may lead to abnormal bleeding. There are many causes ofthrombocytopenia including drug-induced thrombocytopenia (e.g.,following cancer chemotherapy) and immune thromboytopenia (due toincreased degradation of platelets). Platelet transfusions or IL-11 canbe used to restore platelet levels in order to reduce the bleeding risk.

Aplastic Anemia (Pancyteponia)

Aplastic anemia is a life-threatening hematologic disorder characterizedby absent or markedly diminished hematopoietic precursors in the bonemarrow and resulting in neutropenia, anemia and thrombocytopenia. Alarge number of agents can cause aplastic anemia (drugs, chemicals andtoxins) radiation and certain infections can also induce aplasticanemia. More frequently, aplastic anemia occurs as an unpredictableidiosyncratic reaction to drugs such as Antiinflammatory agents,antibiotics, and antiepileptic drugs. Aplastic anemia typically developsweeks or month during drug administration or delayed after drugadministration has been discontinued. Several congenital and familiarforms of aplastic anemia have been described, including Fanconi'sanemia, Shwachman-Diamond syndrome, familiar aplastic anemia, andaplasia associated with dyskeratosis congenita or amegakaryocyticthrompocytopenia.

Cancer

Human MERTK is highly expressed in the following cancer tissues:esophagus tumor, stomach tumor, liver tumor, HEP G2 cells, uterus tumor,ovary tumor, breast tumor, prostate, kidney tumor. The expression in theabove mentioned tissues and in particular the differential expressionbetween diseased tissue and healthy tissue demonstrates that human MERTKprotein or mRNA can be used to diagnose cancer. In addition, theactivity of human MERTK can be modulated to treat cancer.

Cancer is a disease fundamentally caused by oncogenic cellulartransformation. There are several hallmarks of transformed cells thatdistinguish them from their normal counterparts and underlie thepathophysiology of cancer. These include uncontrolled cellularproliferation, unresponsiveness to normal death-inducing signals(immortalization), increased cellular motility and invasiveness,increased ability to recruit blood supply through induction of new bloodvessel formation (angiogenesis), genetic instability, and dysregulatedgene expression. Various combinations of these aberrant physiologies,along with the acquisition of drug-resistance frequently lead to anintractable disease state in which organ failure and patient deathultimately ensue;

Most standard cancer therapies target cellular proliferation and rely onthe differential proliferative capacities between transformed and normalcells for their efficacy. This approach is hindered by the facts thatseveral important normal cell types are also highly proliferative andthat cancer cells frequently become resistant to these agents. Thus, thetherapeutic indices for traditional anti-cancer therapies rarely exceed2.0.

The advent of genomics-driven molecular target identification has openedup the possibility of identifying new cancer-specific targets fortherapeutic intervention that will provide safer, more effectivetreatments for cancer patients. Thus, newly discovered tumor-associatedgenes and their products can be tested for their role(s) in disease andused as tools to discover and develop innovative therapies. Genesplaying important roles in any of the physiological processes outlinedabove can be characterized as cancer targets.

Genes or gene fragments identified through genomics can readily beexpressed in one or more heterologous expression systems to producefunctional recombinant proteins. These proteins are characterized invitro for their biochemical properties and then used as tools inhigh-throughput molecular screening programs to identify chemicalmodulators of their biochemical activities. Agonists and/or antagonistsof target protein activity can be identified in this manner andsubsequently tested in cellular and in vivo disease models foranti-cancer activity. Optimization of lead compounds with iterativetesting in biological models and detailed pharmacokinetic andtoxicological analyses form the basis for drug development andsubsequent testing in humans.

Cancer disorders within the scope of the invention comprise any diseaseof an organ or tissue in mammals characterized by poorly controlled oruncontrolled multiplication of normal or abnormal cells in that tissueand its effect on the body as a whole. Cancer diseases within the Scopeof the invention comprise benign neoplasms, dysplasias, hyperplasias aswell as neoplasms showing metastatic growth or any othertransformations, e.g., leukoplakias, which often precede a breakout ofcancer. Cells and tissues are cancerous when they grow more rapidly thannormal Cells, displacing or spreading into the surrounding healthytissue or any other tis sues of the body described as metastatic growth,assume abnormal shapes and sizes, show changes in theirnucleocytoplasmatic ratio, nuclear polychromasia, and finally may cease.

Cancerous cells and tissues may affect the body as a whole when causingparaneoplastic syndromes or if cancer occurs within a vital organ ortissue, normal function will be impaired or halted, with possible fatalresults. The ultimate involvement of a vital organ by cancer, eitherprimary or metastatic, may lead to the death of the mammal affected.Cancer tends to spread, and the extent of its spread is usually relatedto an individual's chances of surviving the disease. Cancers aregenerally said to be in one of three stages of growth: early, orlocalized, when a tumor is still confined to the tissue of origin, orprimary site; direct extension, where cancer cells from the tumour haveinvaded adjacent tissue or have spread only to regional lymph nodes; ormetastasis, in which cancer cells have migrated to distant parts of thebody from the primary site, via the blood or lymph systems, and haveestablished secondary sites of infection. Cancer is said to be malignantbecause of its tendency to cause death if not treated.

Benign tumors usually do not cause death, although they may if theyinterfere with a normal body function by virtue of their location, size,or paraneoplastic side effects. Hence, benign tumors fall under thedefinition of cancer within the scope of the invention as well. Ingeneral, cancer cells divide at a higher rate than do normal cells, butthe distinction between the growth of cancerous and normal tissues isnot so much the rapidity of cell division in the former as it is thepartial or complete loss of growth restraint in cancer cells and theirfailure to differentiate into a useful, limited tissue of the type thatcharacterizes the functional equilibrium of growth of normal tissue.

Cancer tissues may express certain molecular receptors and probably areinfluenced by the host's susceptibility and immunity and it is knownthat certain cancers of the breast and prostate, for example, areconsidered dependent on specific hormones for their existence. The term“cancer” under the scope of the invention is not limited to simplebenign neoplasia but includes any other benign and malign neoplasia,such as 1) carcinoma, 2) sarcoma, 3) carcinosarcoma, 4) cancers of theblood-forming tissues, 5) tumors of nerve tissues including the brain,and 6) cancer of skin cells.

Carcinoma occurs in epithelial tissues, which cover the outer body (theslin) and line mucous membranes and the inner cavitary structures oforgans e.g. such as the breast, lung, the respiratory andgastrointestinal tracts, the endocrine glands, and the genitourinarysystem. Ductal or glandular elements may persist in epithelial tumors,as in adenocarcinomas, e.g., thyroid adenocarcinoma, gastricadenocarcinoma, uterine adenocarcinoma. Cancers of the pavement-cellepithelium of the skin and of certain mucous membranes, such as cancersof the tongue, lip, larynx, urinary bladder, uterine cervix, or penis,may be termed epidermoid or squamous-cell carcinomas of the respectivetissues and are within the scope of the definition of cancer as well.

Sarcomas develop in connective tissues, including fibrous tissues,adipose (fat) tissues, muscle, blood vessels, bone, and cartilage suchas osteogenic sarcoma, liposarcoma, fibrosarcoma, and synovial sarcoma.

Carcinosarcoma is cancer that develops in both epithelial and connectivetissue. Cancer disease within the scope of this definition may beprimary or secondary, whereby primary indicates that the canceroriginated in the tissue where it is found rather than was establishedas a secondary site through metastasis from another lesion. Cancers andtumor diseases within the scope of this definition may be benign ormalign and may affect all anatomical structures of the body of a mammal.By example, to they comprise cancers and tumor diseases of I) the bonemarrow and bone marrow derived cells (leukemias), II) the endocrine andexocrine glands, such as the thyroid, parathyroid, pituitary, adrenalglands, salivary glands, and pancreas III) the breast, such as benign ormalignant tumors in the mammary glands of either a male or a female, themammary ducts, adenocarcinoma, medullary carcinoma, comedocarcinoma,Paget's disease of the nipple, inflammatory carcinoma of the youngwoman, IV) the lung, V) the stomach, VI) the liver and spleen, VII) thesmall intestine, VIII) the colon, IX) the bone and its supportive andconnective tissues such as malignant or benign bone tumour, such asmalignant osteogenic sarcoma, benign osteoma, cartilage tumors,malignant chondrosarcoma or benign chondroma; bone marrow tumors such asmalignant myeloma or benign eosinophilic granuloma, as well asmetastatic tumors from bone tissues at other locations of the body; X)the mouth, throat, larynx, and the esophagus, XI) the urinary bladderand the internal and external organs and structures of the urogenitalsystem of male and female such as the ovaries, uterus, cervix of theuterus, testes, and prostate gland, XII) the prostate, XIII) thepancreas, such as ductal carcinoma of the pancreas; XII) the lymphatictissue such as lymphomas and other tumors of lymphoid origin, XV) theskin, XVI) cancers and tumor diseases of all anatomical structuresbelonging to the respiratory systems including thoracal muscles andlinings, XVII) primary or secondary cancer of the lymph nodes, XVIII)the tongue and of the bony structures of the hard palate or sinuses,XVII) the mouth, cheeks, neck and salivary glands, XX) the blood vesselsincluding the heart and their linings, XXI) the smooth or skeletalmuscles and their ligaments and linings, XXII) the peripheral, theautonomous, the central nervous system including the cerebellum, andXXIII) the adipose tissue.

Protein Kinases and Cancer Treatment

Protein kinases are a large family of proteins that transfer thegamma-phosphate of ATP to a specific residue(s) of a protein substrate.A protein kinase is classified as a tyrosine, a serine/threonine or adual specific kinase based on the acceptor residue(s). Protein kinasesplay an important role in signaling pathways regulating a number ofcellular functions, such as cell proliferation, apoptosis, angiogenesisand metastasis which are the hallmarks of all cancers. Several proteinkinases have been identified as oncogenes and shown to be dysregulatedin many cancer types, thereby making protein kinases as attractivetherapeutic targets for the treatment of cancer. Drugs targeted againstprotein kinases responsible for the dysregulation of any of theaforementioned pathways have the potential for greater efficacy andlower toxicity. See Kumar & Madison, Expert Opin. Emerging Drugs 6,303-15, 2001.

Signaling pathways for cell growth and proliferation are initiated atthe cell surface by binding of a growth factor ligand to its receptor.Ligand binding to its receptor leads to autophosphorylation andactivation of its tyrosine kinase domain. The growth factor signal isfurther transmitted within a cell through a series of protein kinasesresulting in changes in DNA synthesis, cell cycle, cellular morphology,gene expression, protein translation and metabolic pathways.

Examples of receptor protein tyrosine kinases include EGF receptor, PDGFreceptor, VEGF receptor and FGF receptor. Src, abl and Ick constitutesome of the cytosolic tyrosine kinases. Examples of serine/threoninekinases include MAP kinases, Akt/PKB, and CDKs. Several drugs targetingsome of these protein kinases have reached market (Gleevec) or are inclinical development (Iressa).

Metabolic Disorders

Human MERTK is highly expressed in the following metabolic diseaserelated tissues: liver cirrhosis. The expression in the above mentionedtissues and in particular the differential expression between diseasedtissue and healthy tissue demonstrates that human MERTK protein or mRNAcan be used to diagnose metabolic diseases. In addition, the activity ofhuman MERTK can be modulated to treat metabolic diseases.

Metabolic diseases are defined as conditions that result from anabnormality in any of the chemical or biochemical transformations andtheir regulating systems essential to producing energy, to regeneratingcellular constituents, to eliminating unneeded products arising fromthese processes, and to regulate and maintain homeostasis in a mammalregardless of whether acquired or the result of a genetictransformation. Depending on which metabolic pathway is involved, asingle defective transformation or disturbance of its regulation mayproduce consequences that are narrow, involving a single body function,or broad, affecting many organs, organ systems, or the body as a whole.Diseases resulting from abnormalities related to the fine and coarsemechanisms that affect each individual transformation, its rate anddirection, or the availability of substrates like amino acids, fattyacids, carbohydrates, minerals, cofactors, hormones, regardless whetherthey are inborn or acquired, are well within the scope of the definitionof a metabolic disease according to this application.

Metabolic diseases often are caused by single defects in particularbiochemical pathways, defects that are due to the deficient activity ofindividual enzymes or molecular receptors leading to the regulation ofsuch enzymes. Hence, in a broader sense disturbances of the underlyinggenes, their products and their regulation lie well within the scope ofthis definition of a metabolic disease. For example, metabolic diseasesmay affect 1) biochemical processes and tissues ubiquitous all over thebody, 2) the bone, 3) the nervous system, 4) the endocrine system, 5)the muscle including the heart, 6) the skin and nervous tissue, 7) theurogenital system, 8) the homeostasis of body systems like water andelectrolytes.

Metabolic diseases according to 1) include, but are not limited to,obesity, amyloidosis, disturbances of the amino acid metabolism likebranched chain disease, hyperaminoacidemia, hyperaminoaciduria,disturbances of the metabolism of urea, hyperammonemia,mucopolysaccharidoses (e.g., Maroteaux-Lamy syndrome, storage diseasessuch as glycogen storage diseases and lipid storage diseases,glycogenosis diseases such as Cori's disease, malabsorption diseasessuch as intestinal carbohydrate malabsorption, oligosaccharidasedeficiency like maltase-, lactase-, or sucrase-insufficiency, disordersof the metabolism of fructose, disorders of the metabolism of galactose,galactosemia, disturbances of carbohydrate utilization such as diabetes,hypoglycemia, disturbances of pyruvate metabolism, hypolipidemia,hypolipoproteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine orcarnitine acyltransferase deficiency, disturbances of the porphyrinmetabolism, porphyrias, disturbances of the purine metabolism, lysosomaldiseases, and metabolic diseases of nerves and nervous systems such asgangliosidoses, sphingolipidoses, sulfatidoses, leucodystrophies, andLesch-Nyhan syndrome.

Metabolic diseases according to 2) include, but are not limited to,osteoporosis, osteomalacia-like osteoporosis, osteopenia, osteogenesisimperfecta, osteopetrosis, osteonecrosis, Paget's disease of bone, andhypophosphatemia

Metabolic diseases according to 3) include, but are not limited to,cerebellar dysfunction, disturbances of brain metabolism such asdementia, Alzheimer's disease, Huntington's chorea, Parkinson's disease,Pick's disease, toxic encephalopathy, demyelinating neuropathies such asinflammatory neuropathy, and Guillain-Barré syndrome.

Metabolic diseases according to 4) include, but are not limited to,primary and secondary metabolic disorders associated with hormonaldefects such as any disorder stemming from either a hyperfunction orhypofunction of some hormone-secreting endocrine gland and anycombination thereof. They include Sipple's syndrome, pituitary glanddysfunction and its effects on other endocrine glands, such as thethyroid, adrenals, ovaries, and testes, acromegaly, hyper- andhypothyroidism, euthyroid goiter, euthyroid sick syndrome, thyroiditis,and thyroid cancer, over- or underproduction of the adrenal steroidhormones, adrenogenital syndrome, Cushing's syndrome, Addison's diseaseof the adrenal cortex, Addison's pernicious anemia, primary andsecondary aldosteronism, diabetes insipidus, carcinoid syndrome,disturbances caused by the dysfunction of the parathyroid glands,pancreatic islet cell dysfunction, diabetes, disturbances of theendocrine system of the female such as estrogen deficiency, andresistant ovary syndrome.

Metabolic diseases according to 5) include, but are not limited to,muscle weakness, myotonia, Duchenne's and other muscular dystrophies,dystrophia myotonica of Steinert, mitochondrial myopathies such asdisturbances of the catabolic metabolism in the muscle, carbohydrate andlipid storage myopathies, glycogenoses, myoglobinuria, malignanthyperthermia, polymyalgia rheumatica, dermatomyositis, primarymyocardial disease, cardiomyopathy.

Metabolic diseases according to 6) include, but are not limited to,disorders of the ectoderm, neurofibromatosis, scleroderma andpolyarteritis, Louis-Bar syndrome, von Hippel-Lindau disease,Sturge-Weber syndrome, tuberous sclerosis, amyloidosis, porphyria.

Metabolic diseases according to 7) include, but are not limited to,sexual dysfunction of the male and female.

Metabolic diseases according to 8) include, but are not limited to,confused states and seizures due to inappropriate secretion ofantidiuretic hormone from the pituitary gland, Liddle's syndrome,Bartter's syndrome, Fanconi's syndrome, renal electrolyte wasting,diabetes insipidus.

Diabetes

Diabetes mellitus is a common metabolic disorder characterized by anabnormal elevation in blood glucose, alterations in lipids andabnormalities (complications) in the cardiovascular system, eye, kidneyand nervous system. Diabetes is divided into two separate diseases: type1 diabetes (juvenile onset), which results from a loss of cells whichmake and secrete insulin, and type 2 diabetes (adult onset), which iscaused by a defect in insulin secretion and a defect in insulin action.

Type I diabetes is initiated by an autoimmune reaction that attacks theinsulin secreting cells (beta cells) in the pancreatic islets. Agentsthat prevent this reaction from occurring or that stop the reactionbefore destruction of the beta cells has been accomplished are potentialtherapies for this disease. Other agents that induce beta cellproliferation and regeneration also are potential therapies.

Type II diabetes is the most common of the two diabetic conditions (6%of the population). The defect in insulin secretion is an importantcause of the diabetic condition and results from an inability of thebeta cell to properly detect and respond to rises in blood glucoselevels with insulin release. Therapies that increase the response by thebeta cell to glucose would offer an important new treatment for thisdisease.

The defect in insulin action in Type II diabetic subjects is anothertarget for therapeutic intervention. Agents that increase the activityof the insulin receptor in muscle, liver, and fat will cause a decreasein blood glucose and a normalization of plasma lipids. The receptoractivity can be increased by agents that directly stimulate the receptoror that increase the intracellular signals from the receptor. Othertherapies can directly activate the cellular end process, i.e. glucosetransport or various enzyme systems, to generate an insulin-like effectand therefore a produce beneficial outcome. Because overweight subjectshave a greater susceptibility to Type II diabetes, any agent thatreduces body weight is a possible therapy.

Both Type I and Type diabetes can be treated with agents that mimicinsulin action or that treat diabetic complications by reducing bloodglucose levels. Likewise, agents that reduces new blood vessel growthcan be used to treat the eye complications that develop in bothdiseases.

Neurological Disorders

Human MERTK is highly expressed in the following brain tissues: brain,Alzheimer brain, cerebral cortex, Alzheimer cerebral cortex, frontallobe, Alzheimer brain frontal lobe, occipital lobe, parietal lobe,temporal lobe, precentral gyrus, pons, substantia nigra, corpuscallosum, hippocampus, spinal cord. The expression in brain tissues andin particular the differential expression, between diseased tissue andhealthy tissue demonstrates that human MERTK or mRNA can be utilized todiagnose nervous system diseases. In addition, the activity of humanMERTK can be modulated to treat nervous system diseases.

Central and peripheral nervous system disorders can be treated, such asprimary and secondary disorders after brain injury, disorders of mood,anxiety disorders, disorders of thought and volition, disorders of sleepand wakefulness, diseases of the motor unit, such as neurogenic andmyopathic disorders, neurodegenerative disorders such as Alzheimer's andParkinson's disease, and processes of peripheral and chronic pain.

Pain that is associated with CNS disorders also can be treated byregulating the activity of human receptor tyrosine kinase MerTK. Painwhich can be treated includes that associated with central nervoussystem disorders, such as multiple sclerosis, spinal cord injury,sciatica, failed back surgery syndrome, traumatic brain injury,epilepsy, Parkinson's disease, post-stroke, and vascular lesions in thebrain and spinal cord (e.g., infarct, hemorrhage, vascularmalformation). Non-central neuropathic pain includes that associatedwith post mastectomy pain, reflex sympathetic dystrophy (RSD),trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS relatedpain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, cranial neuralgias, and post-herpeticneuralgia. Pain associated with cancer and cancer treatment also can betreated, as can headache pain (for example, migraine with aura, migrainewithout aura, and other migraine disorders), episodic and chronictension-type headache, tension-type like headache, cluster headache, andchronic paroxysmal hemicrania.

Cardiovascular Disorders

Human MERTK is highly expressed in the following cardiovascular relatedtissues: fetal heart, heart, pericardium, heart atrium (right), heartatrium (left), heart apex, Purkinje fibers, interventricular septum,coronary artery, coronary artery sclerotic, pulmonic valve, HUVEC cells.Expression in the above mentioned tissues and in particular thedifferential expression between diseased tissue and healthy tissuedemonstrates that human MERTK protein or “mRNA can be used to diagnosecardiovascular diseases. In addition, the activity of human MERTK can bemodulated to treat cardiovascular diseases.

Heart failure is defined as a pathophysiological state in which anabnormality of cardiac function is responsible for the failure of theheart to pump blood at a rate commensurate with the requirement of themetabolizing tissue. It includes all forms of pumping failures such ashigh-output and low-output, acute and chronic, right-sided orleft-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease incoronary blood flow that follows a thrombotic occlusion of a coronaryartery previously narrowed by arteriosclerosis. MI prophylaxis (primaryand secondary prevention) is included as well as the acute treatment ofMI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow isrestricted resulting in a perfusion which is inadequate to meet themyocardial requirement for oxygen. This group of diseases includesstable angina, unstable angina and asymptomatic ischemia.

Arrhythmias include all forms of atrial and ventriculartachyarrhythmias, atrial tachycardia, atrial flutter, atrialfibrillation, atrio-ventricular reentrant tachycardia, preexitationsyndrome, ventricular tachycardia, ventricular flutter, ventricularfibrillation, as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds ofsecondary arterial hypertension, renal, endocrine, neurogenic, others.The genes may be used as drug targets for the treatment of hypertensionas well as for the prevention of all complications arising fromcardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in whicharterial and/or venous flow is reduced resulting in an imbalance betweenblood supply and tissue oxygen demand. It includes chronic peripheralarterial occlusive disease (PAOD), acute arterial thrombosis andembolism, inflammatory vascular disorders, Raynaud's phenomenon andvenous disorders.

Atherosclerosis is a cardiovascular disease in which the vessel wall isremodeled, compromising the lumen of the vessel. The atheroscleroticremodeling process involves accumulation of cells, both smooth musclecells and monocyte/macrophage inflammatory cells, in the intima of thevessel wall. These cells take up lipid, likely from the circulation, toform a mature atherosclerotic lesion. Although the formation of theselesions is a chronic process, occurring over decades of an adult humanlife, the majority of the morbidity associated with atherosclerosisoccurs when a lesion ruptures, releasing thrombogenic debris thatrapidly occludes the artery. When such an acute event occurs in thecoronary artery, myocardial infarction can ensue, and in the worst case,can result in death.

The formation of the atherosclerotic lesion can be considered to occurin five overlapping stages such as migration, lipid accumulation,recruitment of inflammatory cells, proliferation of vascular smoothmuscle cells, and extracellular matrix deposition. Each of theseprocesses can be shown to occur in man and in animal models ofatherosclerosis, but the relative contribution of each to the pathologyand clinical significance of the lesion is unclear.

Thus, a need exists for therapeutic methods and agents to treatcardiovascular pathologies, such as atherosclerosis and other conditionsrelated to coronary artery disease.

Cardiovascular diseases include but are not limited to disorders of theheart and the vascular system, such as congestive heart failure,myocardial infarction, ischemic diseases of the heart, all kinds ofatrial and ventricular arrhythmias, hypertensive vascular diseases,peripheral vascular diseases, and atherosclerosis.

Levels of fats in the bloodstream that are too high or too low,especially cholesterol levels, can cause long-term problems. The risk todevelop atherosclerosis and coronary artery or carotid artery disease(and thus the risk of having a heart attack or stroke) increases withthe total cholesterol level increasing. Nevertheless, extremely lowcholesterol levels may not be healthy. Examples of disorders of lipidmetabolism are hyperlipidemia (abnormally high levels of fats(cholesterol, triglycerides, or both) in the blood, may be caused byfamily history of hyperlipidemia, obesity, a high-fat diet, lack ofexercise, moderate to high alcohol consumption, cigarette smoking,poorly controlled diabetes, and an underactive thyroid gland),hereditary hyperlipidemias (type I hyperlipoproteinemia (familialhyperchylomicronemia), type II hyperlipoproteinemia (familialhypercholesterolemia), type III hyperlipoproteinemia, type IVhyperlipoproteinemia, or type V hyperlipoproteinemia),hypolipoproteinemia, lipidoses (caused by abnormalities in the enzymesthat metabolize fats), Gaucher's disease, Niemann-Pick disease, Fabry'sdisease, Wolman's disease, cerebrotendinous xanthomatosis,sitosterolemia, Refsum's disease, or Tay-Sachs disease.

Kidney disorders may lead to hyper or hypotension. Examples for kidneyproblems possibly leading to hypertension are renal artery stenosis,pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidneydisease, injury to the kidney, or radiation therapy affecting thekidney. Excessive urination may lead to hypotension.

Respiratory Disorders

Human MERTK is highly expressed in the following tissues of therespiratory system: leukocytes (peripheral blood), neutrophils cordblood. The expression in the above mentioned tissues demonstrates thathuman MERTK protein or mRNA can be used to diagnose respiratorydiseases. In addition, the activity of human MERTK can be modulated totreat those diseases.

Asthma

Allergy is a complex process in which environmental antigens induceclinically adverse reactions. The inducing antigens, called allergens,typically elicit a specific IgE response and, although in most cases theallergens themselves have little or no intrinsic toxicity, they inducepathology when the IgE response in turn elicits an IgE-dependent or Tcell-dependent hypersensitivity reaction. Hypersensitivity reactions canbe local or systemic and typically occur within minutes of allergenexposure in individuals who have previously been sensitized to anallergen. The hypersensitivity reaction of allergy develops when theallergen is recognized by IgE antibodies bound to specific receptors onthe surface of effector cells, such as mast cells, basophils, oreosinophils, which causes the activation of the effector cells and therelease of mediators that produce the acute signs and symptoms of thereactions. Allergic diseases include asthma, allergic rhinitis (hayfever), atopic dermatitis, and anaphylaxis.

Asthma is thought to arise as a result of interactions between multiplegenetic and environmental factors and is characterized by three majorfeatures: 1) intermittent and reversible airway obstruction caused bybronchoconstriction, increased mucus production, and thickening of thewalls of the airways that leads to a narrowing of the airways, 2) airwayhyperresponsiveness caused by a decreased control of airway caliber, and3) airway inflammation. Certain cells are critical to the inflammatoryreaction of asthma and they include T cells and antigen presentingcells, B cells that produce IgE, and mast cells, basophils, eosinophils,and other cells that bind IgE. These effector cells accumulate at thesite of allergic reaction in the airways and release toxic products thatcontribute to the acute pathology and eventually to the tissuedestruction related to the disorder. Other resident cells, such assmooth muscle cells, lung epithelial cells, mucus-producing cells, andnerve cells may also be abnormal in individuals with asthma and maycontribute to the pathology. While the airway obstruction of asthma,presenting clinically as an intermittent wheeze and shortness of breath,is generally the most pressing symptom of the disease requiringimmediate treatment, the inflammation and tissue destruction associatedwith the disease can lead to irreversible changes that eventually makeasthma a chronic disabling disorder requiring long-term management.

Despite recent important advances in our understanding of thepathophysiology of asthma, the disease appears to be increasing inprevalence and severity (Gergen and Weiss, Am. Rev. Respir. Dis. 146,823-24, 1992). It is estimated that 30-40% of the population suffer withatopic allergy, and 15% of children and 5% of adults in the populationsuffer from asthma (Gergen and Weiss, 1992). Thus, an enormous burden isplaced on our health care resources. However, both diagnosis andtreatment of asthma are difficult. The severity of lung tissueinflammation is not easy to measure and the symptoms of the disease areoften indistinguishable from those of respiratory infections, chronicrespiratory inflammatory disorders, allergic rhinitis, or otherrespiratory disorders. Often, the inciting allergen cannot bedetermined, making removal of the causative environmental agentdifficult. Current pharmacological treatments suffer their own set ofdisadvantages. Commonly used therapeutic agents, such as beta agonists,can act as symptom relievers to transiently improve pulmonary function,but do not affect the underlying inflammation; Agents that can reducethe underlying inflammation, such as anti-inflammatory steroids, canhave major drawbacks that range from immunosuppression to bone loss(Goodman and Gilman's THE PHARMACOLOGIC BASIS OF THERAPEUTICS, SeventhEdition, MacMillan Publishing Company, NY, USA, 1985). In addition, manyof the present therapies, such as inhaled corticosteroids, areshort-lasting, inconvenient to use, and must be used often on a regularbasis, in some cases for life, making failure of patients to comply withthe treatment a major problem and thereby reducing their effectivenessas a treatment.

Because of the problems associated with conventional therapies,alternative treatment strategies have been evaluated. Glycophorin A (Chuand Sharom, Cell. Immunol. 145, 223-39, 1992), cyclosporin (Alexander etal., Lancet 339, 324-28, 1992), and a nonapeptide fragment of IL-2(Zav'yalov et al., Immunol. Lett. 31, 285-88, 1992) all inhibitinterleukin-2 dependent T lymphocyte proliferation; however, they areknown to have many other effects. For example, cyclosporin is used as aimmunosuppressant after organ transplantation. While these agents mayrepresent alternatives to steroids in the treatment of asthmatics, theyinhibit interleukin-2 dependent T lymphocyte proliferation andpotentially critical immune functions associated with homeostasis. Othertreatments that block the release or activity of mediators ofbronchochonstriction, such as cromones or anti-leukotrienes, haverecently been introduced for the treatment of mild asthma, but they areexpensive and not effective in all patients and it is unclear whetherthey have any effect on the chronic changes associated with asthmaticinflammation. What is needed in the art is the identification of atreatment that can act in pathways critical to the development of asthmathat both blocks the episodic attacks of the disorder and preferentiallydampens the hyperactive allergic immune response withoutimmunocompromising the patient.

COPD

Chronic obstructive pulmonary (or airways) disease (COPD) is a conditiondefined physiologically as airflow obstruction that generally resultsfrom a mixture of emphysema and peripheral airway obstruction due tochronic bronchitis (Senior & Shapiro, Pulmonary Diseases and Disorders,3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998; Barnes, Chest117, 10S-14S, 2000). Emphysema is characterized by destruction ofalveolar walls leading to abnormal enlargement of the air spaces of thelung. Chronic bronchitis is defined clinically as the presence ofchronic productive cough for three months in each of two successiveyears. In COPD, airflow obstruction is usually progressive and is onlypartially reversible. By far the most important risk factor fordevelopment of COPD is cigarette smoking, although the disease doesoccur in non-smokers.

Chronic inflammation of the airways is a key pathological feature ofCOPD (Senior & Shapiro, 1998). The inflammatory cell populationcomprises increased numbers of macrophages, neutrophils, and CD8⁺lymphocytes. Inhaled irritants, such as cigarette smoke, activatemacrophages that are resident in the respiratory tract, as well asepithelial cells leading to release of chemokines (e.g., interleukin-8)and other chemotactic factors. These chemotactic factors act to increasethe neutrophil/monocyte trafficking from the blood into the lung tissueand airways. Neutrophils and monocytes recruited into the airways canrelease a variety of potentially damaging mediators such as proteolyticenzymes and reactive oxygen species. Matrix degradation and emphysema,along with airway wall thickening, surfactant dysfunction, and mucushypersecretion, all are potential sequelae of this inflammatory responsethat lead to impaired airflow and gas exchange.

Protein Kinases and Treatment of COPD

Protein kinases are signal transducing enzymes that phosphorylateproteins, including other kinases, and, along with protein phosphatases,regulate the level and extent of protein phosphorylation and activation.Intracellular signalling pathways have important roles in inflammatoryprocesses. These pathways may be activated by cytokines, oxidant stressand other inflammatory mediators (reviewed in Kyraikis & Avruch, J.Biol. Chem. 271, 24313-16, 1996; Kyraikis & Avruch, J. Physiol. Rev. 81,807-69, 2001). For example, the pro-inflammatory cytokines, tumornecrosis factor α (TNFα) and interleukin-1 activate the protein ser/thrkinases c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activatedprotein (MAP) kinase, leading to activation of AP-1 and IKB kinase(IKK), which, in turn, leads to activation of the transcription factorNFKB. Activation of NFKB is required for the transcription of severalproinflammatory molecules, including interleukin-8 and ICAM-1. Enzymesof the MAP kinase class may also act to increase cytokine production bystabilization of mRNA (Winzen et al., EMBO J. 18, 4969980, 1999)

Inhibition of specific protein kinases has been shown to elicitanti-inflammatory effects. For example, the accumulation ofpolymorphonuclear leukocytes in murine lung following intratrachealadministration of bacterial lipopolysaccharide can be blocked byinhibition of p38 MAP kinase (Nick et al., J. Immunol. 164, 2151-59,2000). As a further example, aerosol delivery to rat lungs of antisenseoligodeoxynucleotides to syk kinase mRNA, suppressed nitric oxide andTNFα production from alveolar macrophages stimulated with IgG-anti-IgGcomplexes (Stenton et. al., J. Immunol. 164, 3790-97, 2000). Proteinkinase subtypes are therefore attractive therapeutic targets for theattenuation of the inflammatory response in COPD.

Reproductive Disorders

Human MERTK is highly expressed in the following tissues of thereproduction system: testis, uterus, uterus tumor, ovary, ovary tumor,breast, breast tumor. The expression in the above mentioned tissues andin particular the differential expression between diseased tissue andhealthy tissue demonstrates that human MERTK protein or mRNA can be usedto diagnose reproductive disorders. In addition, the activity of humanMERTK can be modulated to treat reproductive disorders.

Disorders of the male reproductive system include but are not limited tobalanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis,erythroplasia of Queyrat, skin cancer of the penis, Bowen's and Paget'sdiseases, syphilis, herpes simplex infections, genital warts, molluscumcontagiosum, priapism, peyronie's disease, benign prostatic hyperplasia(BPH), prostate cancer, prostatitis, testicular cancer, testiculartorsion, inguinal hernia, epididymo-orchitis, mumps, hydroceles,spermatoceles, or varicoceles. Impotence (erectile dysfunction) mayresults from vascular impairment, neurologic disorders, drugs,abnormalities of the penis, or psychological problems.

Examples of disorders of the female reproductive include prematuremenopause, pelvic pain, vaginitis, vulvitis, vulvovaginitis, pelvicinflammatory disease, fibroids, menstrual disorders (premenstrualsyndrome (PMS), dysmenorrhea, amenorrhea, primary amenorrhea, secondaryamenorrhea, menorrhagia, hypomenorrhea, polymenorrhea, oligomenorrhea,metrorrhagia, menometrorrhagia, Postmenopausal bleeding), bleedingcaused by a physical disorder, dysfunctional uterine bleeding,polycystic ovary syndrome (Stein-Leventhal syndrome), endometriosis,cancer of the uterus, cancer of the cervix, cancer of the ovaries,cancer of the vulva, cancer of the vagina, cancer of the fallopiantubes, and hydatidiform mole.

Infertility may be caused by problems with sperm, ovulation, thefallopian tubes, and the cervix as well as unidentified factors.

Complications of pregnancy include miscarriage and stillbirth, ectopicpregnancy, anemia, Rh incompatibility, problems with the placenta,excessive vomiting, preeclampsia, eclampsia, and skin rashes (e.g.herpes gestationis, urticaria of pregnancy) as well as preterm labor andpremature rupture of the membranes.

Breast disorders may be noncancerous (benign) or cancerous (malignant).Examples of breast disorders are but are not limited to breast pain,cysts, fibrocystic breast disease, fibrous lumps, nipple discharge,breast infection, breast cancer (ductal carcinoma, lobular carcinoma,medullary carcinoma, tubular carcinoma, and inflammatory breast cancer),Paget's disease of the nipple or Cystosarcoma phyllodes.

Endocrine and Hormonal Disorders

Human MERTK is highly expressed in the following tissues of theendocrine system: adrenal gland. The expression in the above mentionedtissues demonstrates that human MERTK protein or mRNA can be used todiagnose endocrine disorders. In addition, the activity of human MERTKcan be modulated to treat endocrine disorders.

The endocrine system consists of a group of organs whose main functionis to produce and secrete hormones directly into the bloodstream. Themajor organs of the endocrine system are the hypothalamus, the pituitarygland, thyroid gland, the parathyroid glands, the islets of thepancreas, the adrenal glands, the testes, and the ovaries. Thehypothalamus secretes several hormones that stimulate the pituitary.Some trigger the release of pituitary hormones, while others suppressthe release of pituitary hormones. The pituitary gland coordinates manyfunctions of the other endocrine glands, but some pituitary hormoneshave direct effects.

The insulin-secreting cells of the pancreas respond to glucose and fattyacids. Parathyroid cells respond to calcium and phosphate. The adrenalmedulla (part of the adrenal gland) responds to direct stimulation bythe parasympathetic nervous system When endocrine glands malfunction,hormone in the blood can become abnormally high or low, disrupting bodyfunctions. Many disorders are caused by malfunction of the endocrinesystem or hormones. Examples of such disorders are presented in thefollowing.

Diabetes mellitus is a disorder in which blood levels of glucose areabnormally high because the body doesn't release or use insulinadequately. People with type I diabetes mellitus (insulin-dependentdiabetes) produce little or no insulin at all. In type I diabetes morethan 90 percent of the insulin-producing cells (beta cells) of thepancreas are permanently destroyed. The resulting insulin deficiency issevere, and to survive, a person with type I diabetes must regularlyinject insulin. In type II diabetes mellitus (non-insulin-dependentdiabetes) the body develops resistance to insulin effects, resulting ina relative insulin deficiency.

The pancreas has two major functions: to secrete fluid containingdigestive enzymes into the duodenum and to secrete the hormones insulinand glucagon. Chronic pancreatitis is a long-standing inflammation ofthe pancreas. Eventually, the insulin-secreting cells of the pancreasmay be destroyed, gradually leading to diabetes. An insulinoma is a raretype of pancreatic tumor that secretes insulin. The symptoms of aninsulinoma result from low blood glucose levels. A gastrinoma is apancreatic tumor that produces excessive levels of the hormone gastrin,which stimulates the stomach to secrete acid and enzymes, causing pepticulcers. The excess gastrin secreted by the gastrinoma causes symptoms,called the Zollinger-Ellison syndrome. A glucagonoma is a tumor thatproduces the hormone glucagon, which raises the level of glucose in theblood and produces a distinctive rash.

Diabetes insipidus is a disorder in which insufficient levels ofantidiuretic hormone cause excessive thirst (polydipsia) and excessiveproduction of very dilute urine (polyuria). Diabetes insipidus resultsfrom the decreased production of antidiuretic hormone (vasopressin).

The body has two adrenal glands. The medulla of the adrenal glandssecretes hormones such as adrenaline (epinephrine) that affect bloodpressure, heart rate, sweating, and other activities also regulated bythe sympathetic nervous system. The cortex secretes many differenthormones, including corticosteroids (cortisone-like hormones), androgens(male hormones), and mineralocorticoids, which control blood pressureand the levels of salt and potassium in the body

A disease characterized by underactive adrenal glands is Addison'sdisease (adreno-cortical insufficiency). Several disorders arecharacterized by overactive Adrenal Glands. The causes can be changes inthe adrenal glands themselves or overstimulation by the pituitary gland.Examples of these diseases are listed in the following. Overproductionof androgenic steroids (testosterone and similar hormones, leads tovirilization), overproduction of corticosteroids (causes could be tumorsof the pituitary or the adrenal gland, results in Cushing's syndrome),Nelson's syndrome (developed by people who have both adrenal glandsremoved, characterized by an enlargement of the pituitary gland),overproduction of aldosterone (hyperaldosteronism), Conn's syndrome(hyperaldosterism caused by a tumor), and pheochromocytoma (a tumor thatoriginating from the adrenal gland's chromaffin cells, causingoverproduction of catecholamines).

The thyroid is a small gland located under the Adam's apple. It secretesthyroid hormones, which control the metabolic rate. The thyroid glandtraps iodine and processes it into thyroid hormones. The euthyroid sicksyndrome is characterized by lack of conversion of the T4 form ofthyroid hormone to the T3 form. Hyperthyroidism (overactive thyroidgland, production of too much hormone) may have several causes.Thyroiditis (an inflammation of the thyroid gland), typically leads to aphase of hyperthyroidism. The inflammation may damage the thyroid gland,so that in later stages the disease is characterized by transient orpermanent underactivity (hypothyroidism). Toxic thyroid nodules(adenomas) often produce thyroid hormone in large quantities. Toxicmultinodular goiter (Plummer's disease) is a disorder in which there aremany nodules. Graves' disease (toxic diffuse goiter) is believed to becaused by an antibody that stimulates the thyroid to produce too muchthyroid hormone. In toxic nodular goiter, one or more nodules in thethyroid produce too much thyroid hormone and aren't under the control ofthyroid-stimulating hormone. Secondary hyperthyroidism may (rarely) becaused by a pituitary tumor that secretes too much thyroid-stimulatinghormone, by resistance of the pituitary to thyroid hormone, whichresults in the pituitary gland secreting too much thyroid-stimulatinghormone, or by a hydatidiform mole in women. Thyroid storm is a suddenextreme overactivity of the thyroid gland is a life-threateningemergency requiring prompt treatment.

Hypothyroidism is a condition in which the thyroid gland is underactiveand produces too little thyroid hormone. Very severe hypothyroidism iscalled myxedema. In Hashimoto's thyroiditis (autoimmune thyroiditis) thethyroid gland is often enlarged and hypothyroidism results because thegland's functioning areas are gradually destroyed. Rarer causes ofhypothyroidism include some inherited disorders that are caused byabnormalities of the enzymes in thyroid cells. In other rare disorders,either the hypothalamus or the pituitary gland fails to secrete enoughof the hormone needed to stimulate normal thyroid function. Otherexamples of thyroiditis are silent lymphocytic thyroiditis, Hashimoto'sthyroiditis, or subacute granulomatous thyroiditis. Thyroid cancer isany one of four main types of malignancy of the thyroid: papillary,follicular, anaplastic, or medullary.

The pituitary is a pea-sized gland that sits in a bony structure (sellaturcica) at the base of the brain. The sella turcica protects thepituitary but allows very little room for expansion. If the pituitaryenlarges, it tends to push upward, often pressing on the areas of thebrain that carry signals from the eyes, possibly resulting in headachesor impaired vision. The pituitary gland has two distinct parts: theanterior (front) and the posterior (back) lobes. The anterior lobeproduces (secretes) hormones that ultimately control the function of thethyroid gland, adrenal glands, and reproductive organs (ovaries andtestes); milk production (lactation) in the breasts; and overall bodygrowth. It also produces hormones that cause the skin to darken and thatinhibit pain sensations. The posterior lobe produces hormones thatregulate water balance, stimulate the let-down of milk from the breastsin lactating women, and stimulate contractions of the uterus.

Examples for disorders of the pituitary gland are Empty Sella Syndrome;hypopituitarism (an underactive pituitary gland); acromegaly, which isexcessive growth caused by oversecretion of growth hormone, which isalmost always caused by a benign pituitary tumor (adenoma);galactorrhea, which is the production of breast milk in men or in womenwho aren't breastfeeding, in both sexes, the most common cause ofgalactorrhea is a prolactin-producing tumor (prolactinoma) in thepituitary gland.

Genitourinary Disorders

Human MERTK is highly expressed in the following urological tissues:prostate, prostate BPH, bladder, ureter, fetal kidney, kidney, kidneytumor. The expression in the above mentioned tissues and in particularthe differential expression between diseased tissue and healthy tissuedemonstrates that human MERTK protein or mRNA can be used to diagnoseurological disorders. In addition, the activity of human MERTK can bemodulated to treat urological disorders.

Genitourological disorders comprise benign and malign disorders of theorgans constituting the genitourological system of female and male,renal diseases like acute or chronic renal failure, immunologicallymediated renal diseases like renal transplant rejection, lupusnephritis, immune complex renal diseases, glomerulopathies, nephritis,toxic nephropathy, obstructive uropathies like benign prostatichyperplasia (BPH), neurogenic bladder syndrome, urinary incontinencelike urge-, stress-, or overflow incontinence, pelvic pain, and erectiledysfunction.

Gastrointestinal and Liver Disorders

Human MERTK is highly expressed in the following tissues of thegastroenterological system: esophagus, esophagus tumor, stomach, stomachtumor, small intestine, rectum, liver, liver cirrhosis, liver tumor, HEPG2 cells. The expression in the above mentioned tissues and inparticular the differential expression between diseased tissue andhealthy tissue demonstrates that human MERTK protein or mRNA can be usedto diagnose gastroenterological disorders. In addition, the activity ofhuman MERTK can be modulated to treat gastroenterological disorders.

Gastrointestinal diseases include primary or secondary, acute or chronicdiseases of the organs of the gastrointestinal tract which may beacquired or inherited, benign or malignant or metaplastic, and which mayaffect the organs of the gastrointestinal tract or the body as a whole.They include but are not limited to 1) disorders of the esophagus suchas achalasia, vigoruos achalasia, dysphagia, cricopharyngealincoordination, pre-esophageal dysphagia, diffuse esophageal spasm,globus sensation, Barrett's metaplasia, gastroesophageal reflux, 2)disorders of the stomach and duodenum such as functional dyspepsia,inflammation of the gastric mucosa, gastritis, stress gastritis, chronicerosive gastritis, atrophy of gastric glands, metaplasia of gastrictissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3)disorders of the pancreas such as acute or chronic pancreatitis,insufficiency of the exocrinic or endocrinic tissues of the pancreassuch as steatorrhea, diabetes, neoplasms of the exocrine or endocrinepancreas such as 3.1) multiple endocrine neoplasia syndrome, ductaladenocarcinoma, cystadenocarcinoma, islet cell tumors, insulinoma,gastrinoma, carcinoid tumors, glucagonoma, Zollinger-Ellison syndrome,Vipoma syndrome, malabsorption syndrome, 4) disorders of the bowel suchas chronic inflammatory diseases of the bowel, Crohn's disease, ileus,diarrhea and constipation, colonic inertia, megacolon, malabsorptionsyndrome, ulcerative colitis, 4.1) functional bowel disorders such asirritable bowel syndrome, 4.2) neoplasms of the bowel such as familialpolyposis, adenocarcinoma, primary malignant lymphoma, carcinoid tumors,Kaposi's sarcoma, polyps, cancer of the colon and rectum.

Liver diseases include primary or secondary, acute or chronic diseasesor injury of the liver which may be acquired or inherited, benign ormalignant, and which may affect the liver or the body as a whole. Theycomprise but are not limited to disorders of the bilirubin metabolism,jaundice, syndromes of Gilbert, Crigler-Najjar, Dubin-Johnson, andRotor; intrahepatic cholestasis, hepatomegaly, portal hyper-tension,ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fattyliver, steatosis, Reye's syndrome, liver diseases due to alcohol,alcoholic hepatitis or cirrhosis, fibrosis and cirrhosis, fibrosis andcirrhosis of the liver due to inborn errors of metabolism or exogenoussubstances, storage diseases, syndromes of Gaucher and Zellweger,Wilson's disease, acute or chronic hepatitis, viral hepatitis and itsvariants; inflammatory conditions of the liver due to viruses, bacteria,fungi, protozoa, helminths; drug induced disorders of the liver, chronicliver diseases such as primary sclerosing cholangitis,alpha1-antitrypsin-deficiency, primary biliary cirrhosis, postoperativeliver disorders such as postoperative intrahepatic cholestasis, hepaticgranulomas, vascular liver disorders associated with systemic disease,benign or malignant neoplasms of the liver, disturbance of livermetabolism in the new-born or prematurely born.

This invention further pertains to the use of novel agents identified bythe screening assays described above. Accordingly, it is within thescope of this invention to use a test compound identified as describedherein in an appropriate animal model. For example, an agent identifiedas described herein (e.g., a modulating agent, an antisense nucleic acidmolecule, a specific antibody, ribozyme, or a human receptor tyrosinekinase MerTK polypeptide binding molecule) can be used in an animalmodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in an animal model to determine the mechanism ofaction of such an agent. Furthermore, this invention pertains to uses ofnovel agents identified by the above-described screening assays fortreatments as described herein.

A reagent which affects receptor tyrosine kinase MerTK activity can beadministered to a human cell, either in vitro or in vivo, to reducereceptor tyrosine kinase MerTK activity. The reagent preferably binds toan expression product of a human receptor tyrosine kinase MerTK gene. Ifthe expression product is a protein, the reagent is preferably anantibody. For treatment of human cells ex vivo, an antibody can be addedto a preparation of stem cells that have been removed from the body. Thecells can then be replaced in the same or another human body, with orwithout clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid compositionthat is capable of fusing with the plasma membrane of the targeted cellto deliver its contents to the cell. Preferably, the transfectionefficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposomedelivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16nmole of liposome delivered to about 10⁶ cells, and even more preferablyabout 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶cells. Preferably, a liposome is between about 100 and 500 nm, morepreferably between about 150 and 450 nm, and even more preferablybetween about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to aparticular cell type, such as a cell-specific ligand exposed on theouter surface of the liposome.

Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods that arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 nmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissuesin vivo using receptor-mediated targeted delivery. Receptor-mediated DNAdelivery techniques are taught in, for example, Findeis et al. Trends inBiotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODSAND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu &Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269,542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59(1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of active ingredient that increases or decreasesenzymatic activity relative to the enzymatic activity which occurs inthe absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model also can be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population), can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors that can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding theantibody can be constructed and introduced into a cell either ex vivo orin vivo using well-established techniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular trasportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAE- orcalcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μgto about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about500 μg/kg of patient body weight, and about 200 to about 250 μg/kg ofpatient body weight. For administration of polynucleotides encodingsingle-chain antibodies, effective in vivo dosages are in the range ofabout 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg ofDNA.

If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides that expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

Preferably, a reagent reduces expression of a human receptor tyrosinekinase MerTK gene or the activity of a receptor tyrosine kinase MerTKpolypeptide by at least about 10, preferably about 50, more preferablyabout 75, 90, or 100% relative to the absence of the reagent. Theeffectiveness of the mechanism chosen to decrease the level ofexpression of a human receptor tyrosine kinase MerTK gene or theactivity of a human receptor tyrosine kinase MerTK polypeptide can beassessed using methods well known in the art, such as hybridization ofnucleotide probes to receptor tyrosine kinase MerTK-specific mRNA,quantitative RT-PCR, immunologic detection of a human receptor tyrosinekinase MerTK polypeptide, or measurement of enzymatic activity.

In any of the embodiments described above, any of the pharmaceuticalcompositions of the invention can be administered in combination withother appropriate therapeutic agents. Selection of the appropriateagents for use in combination therapy can be made by one of ordinaryskill in the art, according to conventional pharmaceutical principles.The combination of therapeutic agents can act synergistically to effectthe treatment or prevention of the various disorders described above.Using this approach, one may be able to achieve therapeutic efficacywith lower dosages of each agent, thus reducing the potential foradverse side effects.

Any of the therapeutic methods described above can be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods

Human receptor tyrosine kinase MerTK also can be used in diagnosticassays for detecting diseases and abnormalities or susceptibility todiseases and abnormalities related to the presence of mutations in thenucleic acid sequences that encode the enzyme. For example, differencescan be determined between the cDNA or genomic sequence encoding receptortyrosine kinase MerTK in individuals afflicted with a disease and innormal individuals. If a mutation is observed in some or all of theafflicted individuals but not in normal individuals, then the mutationis likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene havingmutations can be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments can be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer can beused with a double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures using radiolabeled nucleotides orby automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized, for example, by high resolution gelelectrophoresis. DNA fragments of different sequences can bedistinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985).Sequence changes at specific locations can also be revealed by nucleaseprotection, assays, such as RNase and S 1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85,4397-4401, 1985). Thus, the detection of a specific DNA sequence can beperformed by methods such as hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes andSouthern blotting of genomic DNA. In addition to direct methods such asgel-electrophoresis and DNA sequencing, mutations can also be detectedby in situ analysis.

Altered levels of receptor tyrosine kinase MerTK also can be detected invarious tissues. Assays used to detect levels of the receptorpolypeptides in a body sample, such as blood or a tissue biopsy, derivedfrom a host are well known to those of skill in the art and includeradioimmunoassays, competitive binding assays, Western blot analysis,and ELISA assays.

All patents and patent applications cited in this disclosure areexpressly incorporated herein by reference. The above disclosuregenerally describes the present invention. A more complete understandingcan be obtained by reference to the following specific examples, whichare provided for purposes of illustration only and are not intended tolimit the scope of the invention.

EXAMPLE 1

Detection of Receptor Tyrosine Kinase MerTK Activity

The polynucleotide of SEQ ID NO: 3 is inserted into the expressionvector pCEV4-Myc and the expression vector pCEV4-Myc receptor tyrosinekinase MerTK polypeptide obtained is transfected into human embryonickidney 293 cells. Cells expressing Myc-receptor tyrosine kinase MerTKpolypeptides are identified with anti-Myc-antibody. These cells arecultured in Ham's F-12 medium supplemented with 10% fetal calf serum.The cells are serum-starved and treated with rat or human Gas6 for 10min at 37° C. The cells are rinsed three times with coldphosphate-buffered saline containing 1 mM orthovanadate and lysed withcold lysis buffer. The lysates are immunoprecipitated withanti-Myc-antibodies, run on SDS-PAGE, and immunoblotted withanti-phosphotyrosine monoclonal antibody (PY20). It is shown that thepolypeptide of SEQ ID NO: 2 has a receptor tyrosine kinase MerTKactivity.

EXAMPLE 2

Expression of Recombinant Human Receptor Tyrosine Kinase MerTK

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego,Calif.) is used to produce large quantities of recombinant humanreceptor tyrosine kinase MerTK polypeptides in yeast. The receptortyrosine kinase MerTK-encoding DNA sequence is derived from SEQ ID NO:1.Before insertion into vector pPICZB, the DNA sequence is modified bywell known methods in such a way that it contains at its 5′-end aninitiation codon and at its 3′-end an enterokinase cleavage site, a His6reporter tag and a termination codon. Moreover, at both terminirecognition sequences for restriction endonucleases are added and afterdigestion of the multiple cloning site of pPICZ B with the correspondingrestriction enzymes the modified DNA sequence is ligated into pPICZB.This expression vector is designed for inducible expression in Pichiapastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vectoris used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasksand the recombinantly produced protein isolated from the culture byaffinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea Thebound polypeptide is eluted with buffer, pH 3.5, and neutralized.Separation of the polypeptide from the His6 reporter tag is accomplishedby site-specific proteolysis using enterokinase (Invitrogen, San Diego,Calif.) according to manufacturer's instructions. Purified humanreceptor tyrosine kinase MerTK polypeptide is obtained.

EXAMPLE 3

Identification of Test Compounds that Bind to Receptor Tyrosine KinaseMerTK Polypeptides

Purified receptor tyrosine kinase MerTK polypeptides comprising aglutathione-S-transferase protein and absorbed ontoglutathione-derivatized wells of 96-well microtiter plates are contactedwith test compounds from a small molecule library at pH 7.0 in aphysiological buffer solution. Human receptor tyrosine kinase MerTKpolypeptides comprise the amino acid sequence shown in SEQ ID NO:2. Thetest compounds comprise a fluorescent tag. The samples are incubated for5 minutes to one hour. Control samples are incubated in the absence of atest compound.

The buffer solution containing the test compounds is washed from thewells. Binding of a test compound to a human receptor tyrosine kinaseMerTK polypeptide is detected by fluorescence measurements of thecontents of the wells. A test compound that increases the fluorescencein a well by at least 15% relative to fluorescence of a well in which atest compound is not incubated is identified as a compound which bindsto a human receptor tyrosine kinase MerTK polypeptide.

EXAMPLE 4

Identification of a Test Compound which Decreases Receptor TyrosineKinase MerTK Gene Expression

A test compound is administered to a culture of human cells transfectedwith a receptor tyrosine kinase MerTK expression construct and incubatedat 37° C. for 10 to 45 minutes. A culture of the same type of cells thathave not been transfected is incubated for the same time without thetest compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al.,Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30μg total RNA and hybridized with a ³²P-labeled receptor tyrosine kinaseMerTK-specific probe at 65° C. in Express-hyb (CLONTECH). The probecomprises at least 11 contiguous nucleotides selected from thecomplement of SEQ ID NO:3. A test compound that decreases the receptortyrosine kinase MerTK-specific signal relative to the signal obtained inthe absence of the test compound is identified as an inhibitor ofreceptor tyrosine kinase MerTK gene expression.

EXAMPLE 5

Identification of a Test Compound which Decreases Receptor TyrosineKinase MerTK Activity

Tyrosine receptor kinase MerTK is incubated in 25 μl buffer containing 2mm MnCl₂, 10 mM MgCl₂, 50 mM HEPES (pH 7.6), 150 mM NaCl, 0.1% TRITONX-100, and 0.1% BSA in the presence of 100 nM MerTK ligand and thepresence or absence of a test compound for 1 h at 20° C. A 5 μl mixtureof 1 mg/mL poly(Glu-Tyr) and 10 μM ³²P-ATP is added for 1 h at 20° C.See U.S. Pat. No. 5,939,269. ³²P incorporation is then measured bytrichloroacetic acid precipitability on filter paper. A test compoundwhich decreases the enzymatic activity of the receptor tyrosine kinaserelative to the enzymatic activity in the absence of the test compoundis identified as an inhibitor of receptor tyrosine kinase activity.

EXAMPLE 6

Tissue-Specific Expression of Receptor Tyrosine Kinase MerTK

The qualitative expression pattern of receptor tyrosine kinase MerTK invarious tissues is determined by Reverse Transcription-Polymerase ChainReaction (RT-PCR).

Quantitative Expression Profiling

To demonstrate that receptor tyrosine kinase MerTK is involved incancer, expression is determined in the following tissues: adrenalgland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver,heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate,salivary gland, skeletal muscle, small intestine, spinal cord, spleen,stomach, testis, thymus, thyroid, trachea, uterus, and peripheral bloodlymphocytes. Expression in the following cancer cell lines also isdetermined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205(colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon),MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435(breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignantand normal tissue from the same patient also are tested.

To demonstrate that receptor tyrosine kinase MerTK is involved in thedisease process of diabetes, the following whole body panel is screenedto show predominant or relatively high expression: subcutaneous andmesenteric adipose tissue, adrenal gland, bone marrow, brain, colon,fetal brain, heart, hypothalamus, kidney, liver, lung, mammary gland,pancreas, placenta, prostate, salivary gland, skeletal muscle, smallintestine, spleen, stomach, testis, thymus, thyroid, trachea, anduterus. Human islet cells and an islet cell library also are tested. Asa final step, the expression of receptor tyrosine kinase MerTK in cellsderived from normal individuals with the expression of cells derivedfrom diabetic individuals is compared.

To demonstrate that receptor tyrosine kinase MerTK is involved in CNSdisorders, the following tissues are screened: fetal and adult brain,muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta,trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum,skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala,cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.

To demonstrate that receptor tyrosine kinase MerTK is involved in thedisease process of COPD, the initial expression panel consists of RNAsamples from respiratory tissues and inflammatory cells relevant toCOPD: lung (adult and fetal), trachea, freshly isolated alveolar type IIcells, cultured human bronchial epithelial cells, cultured small airwayepithelial cells, cultured bronchial sooth muscle cells, cultured H441cells (Clara-like), freshly isolated neutrophils and monocytes, andcultured monocytes (macrophage-like). Body map profiling also is carriedout, using total RNA panels purchased from Clontech. The tissues areadrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung,mammary gland, pancreas, prostate, salivary gland, skeletal muscle,small intestine, spleen, stomach, testis, thymus, trachea, thyroid, anduterus.

Quantitative expression profiling is performed by the form ofquantitative PCR analysis called kinetic analysis” firstly described inHiguchi et al., BioTechnology 10, 413-17, 1992, and Higuchi et al.,BioTechnology 11, 1026-30, 1993. The principle is that at any givencycle within the exponential phase of PCR, the amount of product isproportional to the initial number of template copies.

If the amplification is performed in the presence of an internallyquenched fluorescent oligonucleotide (TaqMan probe) complementary to thetarget sequence, the probe is cleaved by the 5′-3′ endonuclease activityof Taq DNA polymerase and a fluorescent dye released in the medium(Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991).Because the fluorescence emission will increase in direct proportion tothe amount of the specific amplified product, the exponential growthphase of PCR product can be detected and used to determine the initialtemplate concentration (Heid et al., Genome Res. 6, 986-94, 1996, andGibson et al., Genome Res. 6, 995-1001, 1996).

The amplification of an endogenous control can be performed tostandardize the amount of sample RNA added to a reaction. In this kindof experiment, the control of choice is the 18S ribosomal RNA. Becausereporter dyes with differing emission spectra are available, the targetand the endogenous control can be independently quantified in the sametube if probes labeled with different dyes are used. All “real time PCR”measurements of fluorescence are made in the ABI Prism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listedabove are used for expression quantification. RNAs labeled “fromautopsy” were extracted from autoptic tissues with the TRIzol reagent(Life Technologies, MD) according to the manufacturer's protocol.

50 μg of each RNA were treated with DNase I for 1 hour at 37° C. in thefollowing reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics,Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mMTris-HCl pH 7.9; 10 mM MgCl₂; 50 mM NaCl; and 1 mM DTT.

After incubation, RNA is extracted once with 1 volume ofphenol:chloro-form:isoamyl alcohol (24:24:1) and once with chloroform,and precipitated with 1/10 volume of 3 M sodium acetate, pH 5.2, and 2volumes of ethanol.

50 μg of each RNA from the autoptic tissues are DNase treated with theDNA-free kit purchased from Ambion (Ambion, Tex.). After resuspensionand spectrophoto-metric quantification, each sample is reversetranscribed with the TaqMan Reverse Transcription Reagents (PE AppliedBiosystems, CA) according to the manufacturer's protocol. The finalconcentration of RNA in the reaction mix is 200 ng/μl. Reversetranscription is carried out with 2.5 μM of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designedaccording to the recommendations of PE Applied Biosystems; the probe canbe labeled at the 5′ end FAM (6-carboxy-fluorescein) and at the 3′ endwith TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experimentsare performed on 10 ng of reverse transcribed RNA from each sample. Eachdetermination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification(multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqManAssay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: 1× final TaqMan Universal PCRMaster Mix (from 2× stock) (PE Applied Biosystems, CA); 1× PDARcontrol—18S RNA (from 20× stock); 300 nM forward primer; 900 nM reverseprimer; 200 nM probe; 10 ng cDNA; and water to 25 μl.

Each of the following steps are carried out once: pre PCR, 2 minutes at50° C., and 10 minutes at 95° C. The following steps are carried out 40times: denaturation, 15 seconds at 95° C., annealing/extension, 1 minuteat 60° C.

The experiment is performed on an ABI Prism 7700 Sequence Detector (PEApplied Biosystems, CA). At the end of the run, fluorescence dataacquired during PCR are processed as described in the ABI Prism 7700user's manual in order to achieve better background subtraction as wellas signal linearity with the starting target quantity.

EXAMPLE 7

Proliferation Inhibition Assay: Antisense Oligonucleotides Suppress theGrowth of Cancer Cell Lines

The cell line used for testing is the human colon cancer cell lineHCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum ata concentration of 10,000 cells per milliliter in a volume of 0.5 ml andkept at 37° C. in a 95% air/5% CO₂ atmosphere.

Phosphorothioate oligoribonucleotides are synthesized on an AppliedBiosystems Model 380B DNA synthesizer using phosphoroamidite chemistry.A sequence of 24 bases complementary to the nucleotides at position 1 to24 of SEQ ID NO:3 is used as the test oligonucleotide. As a control,another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TAC ATG GAC-3′(SEQ ID NO:18). Following assembly and deprotection, oligonucleotidesare ethanol-precipitated twice, dried, and suspended in phosphatebuffered saline at the desired concentration. Purity of theoligonucleotides is tested by capillary gel electrophoresis and ionexchange BPLC. The purified oligonucleotides are added to the culturemedium at a concentration of 10 μM once per day for seven days.

The addition of the test oligonucleotide for seven days results insignificantly reduced expression of human receptor tyrosine kinase MerTKas determined by Western blotting. This effect is not observed with thecontrol oligonucleotide. After 3 to 7 days, the number of cells in thecultures is counted using an automatic cell counter. The number of cellsin cultures treated with the test oligonucleotide (expressed as 100%) iscompared with the number of cells in cultures treated with the controloligonucleotide. The number of cells in cultures treated with the testoligonucleotide is not more than 30% of control, indicating that theinhibition of human receptor tyrosine kinase MerTK has ananti-proliferative effect on cancer cells.

EXAMPLE 8

In Vivo Testing of Compounds/Target Validation for Cancer Treatment

Acute Mechanistic Assays

Reduction in Mitogenic Plasma Hormone Levels

This non-tumor assay measures the ability of a compound to reduce eitherthe endogenous level of a circulating hormone or the level of hormoneproduced in response to a biologic stimulus. Rodents are administeredtest compound (p.o., i.p., i.v., i.m., or s.c.). At a predetermined timeafter administration of test compound, blood plasma is collected. Plasmais assayed for levels of the hormone of interest. If the normalcirculating levels of the hormone are too low and/or variable to provideconsistent results, the level of the hormone may be elevated by apre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.into mice at a dosage of 30 ng/mouse to induce a burst of testosteronesynthesis). The timing of plasma collection would be adjusted tocoincide with the peak of the induced hormone response. Compound effectsare compared to a vehicle-treated control group. An F-test is preformedto determine if the variance is equal or unequal followed by a Student'st-test. Significance is p value≦0.05 compared to the vehicle controlgroup.

Hollow Fiber Mechanism of Action Assay

Hollow fibers are prepared with desired cell line(s) and implantedintraperitoneally and/or subcutaneously in rodents. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol, these may includeassays for gene expression (bDNA, PCR, or Taqman), or a specificbiochemical activity (i.e., cAMP levels. Results are analyzed byStudent's t-test or Rank Sum test after the variance between groups iscompared by an F-test, with significance at p≦0.05 as compared to thevehicle control group.

Subacute Functional In Vivo Assays

Reduction in Mass of Hormone Dependent Tissues

This is another non-tumor assay that measures the ability of a compoundto reduce the mass of a hormone dependent tissue (i.e., seminal vesiclesin males and uteri in females). Rodents are administered test compound(p.o., i.p., i.v., i.m., or s.c.) according to a predetermined scheduleand for a predetermined duration (i.e., 1 week). At termination of thestudy, animals are weighed, the target organ is excised, any fluid isexpressed, and the weight of the organ is recorded. Blood plasma mayalso be collected. Plasma may be assayed for levels of a hormone ofinterest or for levels of test agent. Organ weights may be directlycompared or they may be normalized for the body weight of the animal.Compound effects are compared to a vehicle-treated control group. AnF-test is preformed to determine if the variance is equal or unequalfollowed by a Student's t-test. Significance is p value≦0.05 compared tothe vehicle control group.

Hollow Fiber Proliferation Assay

Hollow fibers are prepared with desired cell line(s) and implantedintraperitoneally and/or subcutaneously in rodents. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol. Cell proliferation isdetermined by measuring a marker of cell number (i.e., MTT or LDH). Thecell number and change in cell number from the starting inoculum areanalyzed by Student's t-test or Rank Sum test after the variance betweengroups is compared by an F-test, with significance at p≦0.05 as comparedto the vehicle control group.

Anti-Angiogenesis Models

Corneal Angiogenesis

Hydron pellets with or without growth factors or cells are implantedinto a micropocket surgically created in the rodent cornea. Compoundadministration may be systemic or local (compound mixed with growthfactors in the hydron pellet). Corneas are harvested at 7 days postimplantation immediately following intracardiac infusion of colloidalcarbon and are fixed in 10% formalin. Readout is qualitative scoringand/or image analysis. Qualitative scores are compared by Rank Sum test.Image analysis data is evaluated by measuring the area ofneovascularization (in pixels) and group averages are compared byStudent's t-test (2 tail). Significance is p≦0.05 as compared to thegrowth factor or cells only group.

Matrigel Angiogenesis

Matrigel, containing cells or growth factors, is injectedsubcutaneously. Compounds are administered p.o., i.p., i.v., i.m., ors.c. Matrigel plugs are harvested at predetermined time point(s) andprepared for readout. Readout is an ELISA-based assay for hemoglobinconcentration and/or histological examination (i.e. vessel count,special staining for endothelial surface markers: CD31, factor-8).Readouts are analyzed by Student's t-test, after the variance betweengroups is compared by an F-test, with significance determined at p≦0.05as compared to the vehicle control group.

Primary Antitumor Efficacy

Early Therapy Models

Subcutaneous Tumor

Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicleand/or compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule starting at a time, usually on Day1, prior to the ability to measure the tumor burden. Body weights andtumor measurements are recorded 2-3 times weekly. Mean net body andtumor weights are calculated for each data collection day. Anti-tumorefficacy may be initially determined by comparing the size of treated(T) and control (C) tumors on a given day by a Student's t-test, afterthe variance between groups is compared by an F-test, with significancedetermined at p≦0.05. The experiment may also be continued past the endof dosing in which case tumor measurements would continue to be recordedto monitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p≦0.05.

Intraperitoneal/Intracranial Tumor Models

Tumor cells are injected intraperitoneally or intracranially on Day 0.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule starting on Day 1. Observations of morbidityand/or mortality are recorded twice daily. Body weights are measured andrecorded twice weekly. Morbidity/mortality data is expressed in terms ofthe median time of survival and the number of long-term survivors isindicated separately. Survival times are used to generate Kaplan-Meiercurves. Significance is p≦0.05 by a log-rank test compared to thecontrol group in the experiment.

Established Disease Model

Tumor cells or fragments are implanted subcutaneously and grown to thedesired size for treatment to begin. Once at the predetermined sizerange, mice are randomized into treatment groups. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. according to apredetermined schedule. Tumor and body weights are measured and recorded2-3 times weekly. Mean tumor weights of all groups over days postinoculation are graphed for comparison. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group.

Orthotopic Disease Models

Mammary Fat Pad Assay

Tumor cells or fragments, of mammary adenocarcinoma origin, areimplanted directly into a surgically exposed and reflected mammary fatpad in rodents. The fat pad is placed back in its original position andthe surgical site is closed. Hormones may also be administered to therodents to support the growth of the tumors. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.Tumor and body weights are measured and recorded 2-3 times weekly. Meantumor weights of all groups over days post inoculation are graphed forcomparison. An F-test is preformed to determine if the variance is equalor unequal followed by a Student's t-test to compare tumor sizes in thetreated and control groups at the end of treatment. Significance isp≦0.05 as compared to the control group.

Tumor measurements may be recorded after dosing has stopped to monitortumor growth delay. Tumor growth delays are expressed as the differencein the median time for the treated and control groups to attain apredetermined size divided by the median time for the control group toattain that size. Growth delays are compared by generating Kaplan-Meiercurves from the times for individual tumors to attain the evaluationsize. Significance is p value≦0.05 compared to the vehicle controlgroup. In addition, this model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ, or measuring the target organ weight. The meansof these endpoints are compared by Student's t-test after conducting anF-test, with significance determined at p≦0.05 compared to the controlgroup in the experiment.

Intraprostatic Assay

Tumor cells or fragments, of prostatic adenocarcinoma origin, areimplanted directly into a surgically exposed dorsal lobe of the prostatein rodents. The prostate is externalized through an abdominal incisionso that the tumor can be implanted specifically in the dorsal lobe whileverifying that the implant does not enter the seminal vesicles. Thesuccessfully inoculated prostate is replaced in the abdomen and theincisions through the abdomen and skin are closed. Hormones may also beadministered to the rodents to support the growth of the tumors.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., thelungs), or measuring the target organ weight (i.e., the regional lymphnodes). The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

Intrabronchial Assay

Tumor cells of pulmonary origin may be implanted intrabronchially bymaking an incision through the skin and exposing the trachea. Thetrachea is pierced with the beveled end of a 25 gauge needle and thetumor cells are inoculated into the main bronchus using a flat-ended 27gauge needle with a 90° bend. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Body weightsare measured and recorded 2-3 times weekly. At a predetermined time, theexperiment is terminated and the animal is dissected. The size of theprimary tumor is measured in three dimensions using either a caliper oran ocular micrometer attached to a dissecting scope. An F-test ispreformed to determine if the variance is equal or unequal followed by aStudent's t-test to compare tumor sizes in the treated and controlgroups at the end of treatment. Significance is p≦0.05 as compared tothe control group. This model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ (i.e., the contralateral lung), or measuring thetarget organ weight. The means of these endpoints are compared byStudent's t-test after conducting an F-test, with significancedetermined at p≦0.05 compared to the control group in the experiment.

Intracecal Assay

Tumor cells of gastrointestinal origin may be implanted intracecally bymaking an abdominal incision through the skin and externalizing theintestine. Tumor cells are inoculated into the cecal wall withoutpenetrating the lumen of the intestine using a 27 or 30 gauge needle.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., theliver), or measuring the target organ weight. The means of theseendpoints are compared by Student's t-test after conducting an F-test,with significance determined at p≦0.05 compared to the control group inthe experiment.

Secondary (Metastatic) Antitumor Efficacy

Spontaneous Metastasis

Tumor cells are inoculated s.c. and the tumors allowed to grow to apredetermined range for spontaneous metastasis studies to the lung orliver. These primary tumors are then excised. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedulewhich may include the period leading up to the excision of the primarytumor to evaluate therapies directed at inhibiting the early stages oftumor metastasis. Observations of morbidity and/or mortality arerecorded daily. Body weights are measured and recorded twice weekly.Potential endpoints include survival time, numbers of visible foci pertarget organ, or target organ weight. When survival time is used as theendpoint the other values are not determined. Survival data is used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment. The mean number ofvisible tumor foci, as determined under a dissecting microscope, and themean target organ weights are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment for both of these endpoints.

Forced Metastasis

Tumor cells are injected into the tail vein, portal vein, or the leftventricle of the heart in experimental (forced) lung, liver, and bonemetastasis studies, respectively. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Observationsof morbidity and/or mortality are recorded daily. Body weights aremeasured and recorded twice weekly. Potential endpoints include survivaltime, numbers of visible foci per target organ, or target organ weight.When survival time is used as the endpoint the other values are notdetermined. Survival data is used to generate Kaplan-Meier curves.Significance is p≦0.05 by a log-rank test compared to the control groupin the experiment. The mean number of visible tumor foci, as determinedunder a dissecting microscope, and the mean target organ weights arecompared by Student's t-test after conducting an F-test, withsignificance at p≦0.05 compared to the vehicle control group in theexperiment for both endpoints.

EXAMPLE 9

Diabetes: In Vivo Testing of Compounds/Target Validation

Glucose Production

Over-production of glucose by the liver, due to an enhanced rate ofgluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes (p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

Insulin Secretion

Compounds that enhance insulin secretion from the pancreas will increaseplasma insulin levels and improve the disappearance of plasma glucosefollowing the administration of a glucose load. When measuring insulinlevels, compounds are administered by different routes (p.o., i.p., s.c.or i.v.) to overnight fasted normal rats or mice. At the appropriatetime an intravenous glucose load (0.4 g/kg) is given, blood is collectedone minute later. Plasma insulin levels are determined. Compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60 and 90 minutes and plasma glucose levelsdetermined. Compounds that increase insulin levels will decrease glucoselevels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

Compounds that enhance insulin secretion from the pancreas will increaseplasma insulin levels and improve the disappearance of plasma glucosefollowing the administration of a glucose load. When measuring insulinlevels, test compounds which regulate receptor tyrosine kinase MerTK areadministered by different routes (p.o., i.p., s.c., or i.v.) toovernight fasted normal rats or mice. At the appropriate time anintravenous glucose load (0.4 g/kg) is given, blood is collected oneminute later. Plasma insulin levels are determined. Test compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60, and 90 minutes and plasma glucose levelsdetermined. Test compounds that increase insulin levels will decreaseglucose levels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

EXAMPLE 10

In Vivo Testing of Compounds/Target Validation for the Treatment ofNeurological Disorders

Pain

Acute pain. Acute pain is measured on a hot plate mainly in rats. Twovariants of hot plate testing are used: In the classical variant animalsare put on a hot surface (52 to 56° C.) and the latency time is measureduntil the animals show nocifensive behavior, such as stepping or footlicking. The other variant is an increasing temperature hot plate wherethe experimental animals are put on a surface of neutral temperature.Subsequently this surface is slowly but constantly heated until theanimals begin to lick a hind paw. The temperature which is reached whenhind paw licking begins is a measure for pain threshold.

Compounds are tested against a vehicle treated control group. Substanceapplication is performed at different time points via differentapplication routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal,transdermal) prior to pain testing.

Persistent pain. Persistent pain is measured with the formalin orcapsaicin test, mainly in rats. A solution of 1 to 5% formalin or 10 to100 μg capsaicin is injected into one hind paw of the experimentalanimal. After formalin or capsaicin application the animals shownocifensive reactions like flinching, licking and biting of the affectedpaw. The number of nocifensive reactions within a time frame of up to 90minutes is a measure for intensity of pain.

Compounds are tested against a vehicle treated control group. Substanceapplication is performed at different time points via differentapplication routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal,transdermal) prior to formalin or capsaicin administration.

Neuropathic pain. Neuropathic pain is induced by different variants ofunilateral sciatic nerve injury mainly in rats. The operation isperformed under anesthesia The first variant of sciatic nerve injury isproduced by placing loosely constrictive ligatures around the commonsciatic nerve. The second variant is the tight ligation of about thehalf of the diameter of the common sciatic nerve. In the next variant, agroup of models is used in which tight ligations or transections aremade of either the L5 and L6 spinal nerves, or the L % spinal nerveonly. The fourth variant involves an axotomy of two of the threeterminal branches of the sciatic nerve (tibial and common peronealnerves) leaving the remaining sural nerve intact whereas the lastvariant comprises the axotomy of only the tibial branch leaving thesural and common nerves uninjured. Control animals are treated with asham operation.

Postoperatively, the nerve injured animals develop a chronic mechanicalallodynia, cold allodynioa, as well as a thermal hyperalgesia.Mechanical allodynia is measured by means of a pressure transducer(electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA; Electronic von Frey System,Somedic Sales AB, Hörby, Sweden). Thermal hyperalgesia is measured bymeans of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy), or by means of a cold plate of 5 to 10° C. where the nocifensivereactions of the affected hind paw are counted as a measure of painintensity. A further test for cold induced pain is the counting ofnocifensive reactions, or duration of nocifensive responses afterplantar administration of acetone to the affected hind limb. Chronicpain in general is assessed by registering the circadanian rhythms inactivity (Surjo and Arndt, Universitat zu Köln, Cologne, Germany), andby scoring differences in gait (foot print patterns; FOOTPRINTS program,Klapdor et al., 1997. A low cost method to analyze footprint patterns.J. Neurosci. Methods 75, 49-54).

Compounds are tested against sham operated and vehicle treated controlgroups. Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to pain testing.

Inflammatory Pain. Inflammatory pain is induced mainly in rats byinjection of 0.75 mg carrageenan or complete Freund's adjuvant into onehind paw. The animals develop an edema with mechanical allodynia as wellas thermal hyperalgesia Mechanical allodynia is measured by means of apressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-LifeScience Instruments, Woodland Hills, SA, USA). Thermal hyperalgesia ismeasured by means of a radiant heat source (Plantar Test, Ugo Basile,Comerio, Italy, Paw thermal stimulator, G. Ozaki, University ofCalifornia, USA). For edema measurement two methods are being used. Inthe first method, the animals are sacrificed and the affected hindpawssectioned and weighed. The second method comprises differences in pawvolume by measuring water displacement in a plethysmometer (Ugo Basile,Comerio, Italy).

Compounds are tested against uninflamed as well as vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,s.c., intradermal, transdermal) prior to pain testing.

Diabetic neuropathic pain. Rats treated with a single intraperitonealinjection of 50 to 80 mg/kg streptozotocin develop a profoundhyperglycemia and mechanical allodynia within 1 to 3 weeks. Mechanicalallodynia is measured by means of a pressure transducer (electronic vonFrey Anesthesiometer, IITC Inc.-Life Science Instruments, WoodlandHills, SA, USA).

Compounds are tested against diabetic and non-diabetic vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,s.c., intradermal, transdermal) prior to pain testing.

Parkinson's Disease

6-Hydroxydopamine (6-OH-DA) Lesion. Degeneration of the dopaminergicnigrostriatal and striatopallidal pathways is the central pathologicalevent in Parkinson's disease. This disorder has been mimickedexperimentally in rats using single/sequential unilateral stereotaxicinjections of 6-OH-DA into the medium forebrain bundle (MFB).

Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 g at thebegining of the experiment, are used. The rats are maintained in atemperature- and humidity-controlled environment under a 12 h light/darkcycle with free access to food and water when not in experimentalsessions. The following in vivo protocols are approved by thegovernmental authorities. All efforts are made to minimize animalsuffering, to reduce the number of animals used, and to utilizealternatives to in vivo techniques.

Animals are administered pargyline on the day of surgery (Sigma, St.Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of 6-OHDAby monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kg i.p.)in order to prevent uptake of 6-OHDA by noradrenergic terminals. Thirtyminutes later the rats are anesthetized with sodium pentobarbital (50mg/kg) and placed in a stereotaxic frame. In order to lesion the DAnigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μgof 6-OHDA HBr (Sigma) are injected into the left medial fore-brainbundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, −2.7 mmventral to Bregma and the skull surface). The needle is left in place anadditional 5 min to allow diffusion to occur.

Stepping Test. Forelimb akinesia is assessed three weeks followinglesion placement using a modified stepping test protocol. In brief, theanimals are held by the experimenter with one hand fixing the hindlimbsand slightly raising the hind part above the surface. One paw istouching the table, and is then moved slowly sideways (5 s for 1 m),first in the forehand and then in the backhand direction. The number ofadjusting steps is counted for both paws in the backhand and forehanddirection of movement. The sequence of testing is right paw forehand andbackhand adjusting stepping, followed by left paw forehand and backhanddirections. The test is repeated three times on three consecutive days,after an initial training period of three days prior to the firsttesting. Forehand adjusted stepping reveals no consistent differencesbetween lesioned and healthy control animals. Analysis is thereforerestricted to backhand adjusted stepping.

Balance Test. Balance adjustments following postural challenge are alsomeasured during the stepping test sessions. The rats are held in thesame position as described in the stepping test and, instead of beingmoved sideways, tilted by the experimenter towards the side of the pawtouching the table. This maneuver results in loss of balance and theability of the rats to regain balance by forelimb movements is scored ona scale ranging from 0 to 3. Score 0 is given for a normal forelimbplacement. When the forelimb movement is delayed but recovery ofpostural balance detected, score 1 is given. Score 2 represents a clear,yet insufficient, forelimb reaction, as evidenced by muscle contraction,but lack of success in recovering balance, and score 3 is given for noreaction of movement. The test is repeated three times a day on eachside for three consecutive days after an initial training period ofthree days prior to the first testing.

Staircase Test (Paw Reaching). A modified version of the staircase testis used for evaluation of paw reaching behavior three weeks followingprimary and secondary lesion placement. Plexiglass test boxes with acentral platform and a removable staircase on each side are used. Theapparatus is designed such that only the paw on the same side at eachstaircase can be used, thus providing a measure of independent forelimbuse. For each test the animals are left in the test boxes for 15 min.The double staircase is filled with 7×3 chow pellets (Precision foodpellets, formula: P, purified rodent diet, size 45 mg; SandownScientific) on each side. After each test the number of pellets eaten(successfully retrieved pellets) and the number of pellets taken(touched but dropped) for each paw and the success rate (pelletseaten/pellets taken) are counted separately. After three days of fooddeprivation (12 g per animal per day) the animals are tested for 11days. Full analysis is conducted only for the last five days.

MPTP treatment. The neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes degenerationof mesencephalic dopaminergic (DAergic) neurons in rodents, non-humanprimates, and humans and, in so doing, reproduces many of the symptomsof Parkinson's disease. MPTP leads to a marked decrease in the levels ofdopamine and its metabolites, and in the number of dopaminergicterminals in the striatum as well as severe loss of the tyrosinehydroxylase (TH)-immunoreactive cell bodies in the substantia nigra,pars compacta.

In order to obtain severe and long-lasting lesions, and to reducemortality, animals receive single injections of MPTP, and are thentested for severity of lesion 7-10 days later. Successive MPTPinjections are administered on days 1, 2 and 3. Animals receiveapplication of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily.All injections are intraperitoneal (i.p.) and the MPTP stock solution isfrozen between injections. Animals are decapitated on day 11.

Immunohistology. At the completion of behavioral experiments, allanimals are anaesthetized with 3 ml thiopental (1 g/40 ml i.p., TyrolPharma). The mice are perfused transcardially with 0.01 M PBS (pH 7.4)for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min.The brains are removed and placed in 4% paraformaldehyde for 24 h at 4°C. For dehydration they are then transferred to a 20% sucrose (Merck)solution in 0.1 M PBS at 4° C. until they sink. The brains are frozen inmethylbutan at −20° C. for 2 min and stored at −70° C. Using a sledgemicrotome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from thegenu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm)and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored inassorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.

A series of sections is processed for free-floating tyrosine hydroxylase(TH) immunohistochemistry. Following three rinses in 0.1 M PBS,endogenous peroxidase activity is quenched for 10 min in 0.3% H₂O₂±PBS.After rinsing in PBS, sections are preincubated in 10% normal bovineserum (Sigma) for 5 min as blocking agent and transferred to eitherprimary anti-rat TH rabbit antiserum (dilution 1:2000).

Following overnight incubation at room temperature, sections for THimmuno-reactivity are rinsed in PBS (2×10 min) and incubated inbiotinylated anti-rabbit immunoglobulin G raised in goat (dilution1:200) (Vector) for 90 min, rinsed repeatedly and transferred toVectastain ABC (Vector) solution for 1 h. 3,3′-Diaminobenzidinetetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005%H₂O₂ serves as chromogen in the subsequent visualization reaction.Sections are mounted on to gelatin-coated slides, left to dry overnight,counter-stained with hematoxylin dehydrated in ascending alcoholconcentrations and cleared in butylacetate. Coverslips are mounted onentellan.

Rotarod Test. We use a modification of the procedure described by Rozasand Labandeira-Garcia (1997), with a CR-1 Rotamex system (ColumbusInstruments, Columbus, Ohio) comprising an IBM-compatible personalcomputer, a CIO-24 data acquisition card, a control unit, and afour-lane rotarod unit. The rotarod unit consists of a rotating spindle(diameter 7.3 cm) and individual compartments for each mouse. The systemsoftware allows preprogramming of session protocols with varyingrotational speeds (0-80 rpm). Infrared beams are used to detect when amouse has fallen onto the base grid beneath the rotarod. The system logsthe fall as the end of the experiment for that mouse, and the total timeon the rotarod, as well as the time of the fall and all the set-upparameters, are recorded. The system also allows a weak current to bepassed through the base grid, to aid training.

Dementia

The object recognition task. The object recognition task has beendesigned to assess the effects of experimental manipulations on thecognitive performance of rodents. A rat is placed in an open field, inwhich two identical objects are present. The rats inspects both objectsduring the first trial of the object recognition task. In a secondtrial, after a retention interval of for example 24 hours, one of thetwo objects used in the first trial, the ‘familiar’ object, and a novelobject are placed in the open field. The inspection time at each of theobjects is registered. The basic measures in the OR task is the timespent by a rat exploring the two object the second trial. Good retentionis reflected by higher exploration times towards the novel than the‘familiar’ object.

Administration of the putative cognition enhancer prior to the firsttrial predominantly allows assessment of the effects on acquisition, andeventually on consolidation processes. Administration of the testingcompound after the first trial allows to assess the effects onconsolidation processes, whereas administration before the second trialallows to measure effects on retrieval processes.

The passive avoidance task. The passive avoidance task assesses memoryperformance in rats and mice. The inhibitory avoidance apparatusconsists of a two-compartment box with a light compartment and a darkcompartment. The two compartments are separated by a guillotine doorthat can be operated by the experimenter. A threshold of 2 cm separatesthe two compartments when the guillotine door is raised. When the dooris open, the illumination in the dark compartment is about 2 lux. Thelight intensity is about 500 lux at the center of the floor of the lightcompartment.

Two habituation sessions, one shock session, and a retention session aregiven, separated by inter-session intervals of 24 hours. In thehabituation sessions and the retention session the rat is allowed toexplore the apparatus for 300 sec. The rat is placed in the lightcompartment, facing the wall opposite to the guillotine door. After anaccommodation period of 15 sec. the guillotine door is opened so thatall parts of the apparatus can be visited freely. Rats normally avoidbrightly lit areas and will enter the dark compartment within a fewseconds.

In the shock session the guillotine door between the compartments islowered as soon as the rat has entered the dark compartment with itsfour paws, and a scrambled 1 mA footshock is administered for 2 sec. Therat is removed from the apparatus and put back into its home cage. Theprocedure during the retention session is identical to that of thehabituation sessions.

The step-through latency, that is the first latency of entering the darkcompartment (in sec.) during the retention session is an index of thememory performance of the animal; the longer the latency to enter thedark compartment, the better the retention is. A testing compound ingiven half an hour before the shock session, together with 1 mg*kg⁻¹scopolamine. Scopolamine impairs the memory performance during theretention session 24 hours later. If the test compound increases theenter latency compared with the scopolamine-treated controls, is likelyto possess cognition enhancing potential.

The Morris water escape task. The Morris water escape task measuresspatial orientation learning in rodents. It is a test system that hasextensively been used to investigate the effects of putative therapeuticon the cognitive functions of rats and mice. The performance of ananimal is assessed in a circular water tank with an escape platform thatis submerged about 1 cm below the surface of the water. The escapeplatform is not visible for an animal swimming in the water tank.Abundant extra-maze cues are provided by the furniture in the room,including desks, computer equipment, a second water tank, the presenceof the experimenter, and by a radio on a shelf that is playing softly.

The animals receive four trials during five daily acquisition sessions.A trial is started by placing an animal into the pool, facing the wallof the tank. Each of four starting positions in the quadrants north,east, south, and west is used once in a series of four trials; theirorder is randomized. The escape platform is always in the same position.A trial is terminated as soon as the animal had climbs onto the escapeplatform or when 90 seconds have elapsed, whichever event occurs first.The animal is allowed to stay on the platform for 30 seconds. Then it istaken from the platform and the next trial is started. If an animal didnot find the platform within 90 seconds it is put on the platform by theexperimenter and is allowed to stay there for 30 seconds. After thefourth trial of the fifth daily session, an additional trial is given asa probe trial: the platform is removed, and the time the animal spendsin the four quadrants is measured for 30 or 60 seconds. In the probetrial, all animals start from the same start position, opposite to thequadrant where the escape platform had been positioned duringacquisition.

Four different measures are taken to evaluate the performance of ananimal during acquisition training: escape latency, traveled distance,distance to platform, and swimming speed. The following measures areevaluated for the probe trial: time (s) in quadrants and traveleddistance (cm) in the four quadrants. The probe trial provides additionalinformation about how well an animal learned the position of the escapeplatform. If an animal spends more time and swims a longer distance inthe quadrant where the platform had been positioned during theacquisition sessions than in any other quadrant, one concludes that theplatform position has been learned well.

In order to assess the effects of putative cognition enhancingcompounds, rats or mice with specific brain lesions which impaircognitive functions, or animals treated with compounds such asscopolamine or MK-801, which interfere with normal learning, or agedanimals which suffer from cognitive deficits, are used.

The T-maze spontaneous alternation task. The T-maze spontaneousalternation task (TeMCAT) assesses the spatial memory performance inmice. The start arm and the two goal arms of the T-maze are providedwith guillotine doors which can be operated manually by theexperimenter. A mouse is put into the start arm at the beginning oftraining. The guillotine door is closed. In the first trial, the ‘forcedtrial’, either the left or right goal arm is blocked by lowering theguillotine door. After the mouse has been released from the start arm,it will negotiate the maze, eventually enter the open goal arm, andreturn to the start position, where it will be confined for 5 seconds,by lowering the guillotine door. Then, the animal can choose freelybetween the left and right goal arm (all guillotine-doors opened) during14 ‘free choice’ trials. As soon a the mouse has entered one goal arm,the other one is closed. The mouse eventually returns to the start armand is free to visit whichever go alarm it wants after having beenconfined to the start arm for 5 seconds. After completion of 14 freechoice trials in one session, the animal is removed from the maze.During training, the animal is never handled.

The percent alternations out of 14 trials is calculated. This percentageand the total time needed to complete the first forced trial and thesubsequent 14 free choice trials (in s) is analyzed. Cognitive deficitsare usually induced by an injection of scopolamine, 30 min before thestart of the training session. Scopolamine reduced the per-centalternations to chance level, or below. A cognition enhancer, which isalways administered before the training session, will at leastpartially, antagonize the scopolamine-induced reduction in thespontaneous alternation rate.

EXAMPLE 11

Identification of Test Compound Efficacy in an Animal Model of COPD

A/J mice are exposed to the smoke from 2 unfiltered cigarettes per dayfor 6 days per week for 14 weeks. Non-smoking, age-matched animals areused as controls. Animals are orally dosed with test compound or vehicle1 hour before and 7 hours after smoke exposure. This twice-daily dosingregime is continued throughout the smoke exposure period. On day 7 ofthe weekly exposure, animals are given only 1 dose of test compound andare not exposed to cigarette smoke.

After the smoke exposure period, the mice are killed, their lungsinflated with phosphate-buffered formalin via their trachea, and thenthe lungs and heart are removed en bloc and fixed at 4° C. for 48 hours.The lungs are then prepared for paraffin wax sectioning, and 4 mmsections are cut and mounted on glass slides. Sections are then stainedwith haematoxylin and eosin. Morphometric analysis of lung sections isdone by calculation of the Linear Mean Intercept (LMI) parameter using asemi-automated computer image analysis system. Each slide (1 per mouse)contains several sections originating from multiple lobes. Twelvenon-overlapping areas (each area covering 1.53×10-3 cm²) are randomlyselected for LMI analysis. The 12 areas cover a minimum of two lobes perslide. Non-parenchymal components (airways, blood vessels) are excludedfrom the analysis to prevent artifactual error. The mean interceptlength is calculated for each mouse. Development of emphysema is seen asan increase in LMI.

LMI data are expressed as the median and statistical comparisons aredone using the non-parametric Mann-Witney U-test. A ‘p’ value of <=0.05is considered to be statistically significant. The potency of a testcompound is evaluated by comparison of the tobacco smoke inducedincrease in LMI in animals dosed with either the test compound or justthe vehicle used for administration of the compound.

EXAMPLE 12

Identification of Test Compound Efficacy in an In Vitro Functional TestRelevant to COPD

The potency of test compounds is evaluated by measuring the inhibitionof elastolysis induced by human alveolar macrophages. The cells areisolated from bronchoalveolar lavage samples taken from non-smokers,disease-free smokers, and smokers with COPD. Macrophage suspensions areadded to test wells coated with tritiated elastin and incubated at 37°C. for 3 h to allow adherence of the cells. The wells are then carefullywashed to remove non-adherent cells and fresh medium is added to eachwell. The cells are incubated at 37° C. for up to 72 hours in thepresence or absence of test compound. Every 24 hours the medium in eachwell is removed for analysis and replaced by fresh medium. Radioactivityreleased into the medium is measured by liquid scintillation countingand the rate of elastin degradation is calculated. The potency of a testcompound is evaluated by comparing the rate of elastolysis measured withcells incubated in the presence or absence of the compound.

EXAMPLE 13

In Vivo Target Validation for the Treatment of Atherosclerosis

Effects on plasma cholesterol levels including HDL cholesterol aretypically assessed in humanized apo-AI transgenic mice. Modulation ofhuman target proteins can be determined in corresponding transgenic mice(e.g., CETP transgenic mice). Triglyceride-lowering is usually evaluatedin ob/ob mice or Zucker rats. Animals are fed with normal diets ormodified diets (e.g., enriched by 0.5% cholesterol 20% coconut oil).Standard protocols consist of oral applications once daily for 7 to 10days at doses ranging from 0,1 to 100 mg/kg. The compounds are dissolved(e.g., in Solutol/Ethanol/saline mixtures) and applied by oral gavage orintravenous injection. Before and at the end of the application period,blood samples are typically drawn by retroorbital punctuation. Plasmacholesterol and triglyceride levels are determined with standardizedclinical diagnostic kits (e.g., INFINITY™ cholesterol reagent andINFINITY™ triglyceride reagent; Sigma, St. Louis). HDL cholesterol isdetermined after phosphotungstic acid precipitation of non-HDLlipoproteins or FPLC gel filtration with post-column derivatization ofcholesterol using the reagents mentioned above. Plasma levels of humanapolipoprotein-AI in relevant humanized transgenic mice are measured byimmunoturbidimetry (Sigma).

Long-term anti-atherosclerotic potency of drug candidates are evaluatedin Apo E-knockout mice. Therefore, animals are fed a standard chow diet(4,5% fat) or a Western diet (20% fat) containing 1 to 100 mg/kg of therespective compounds for 3 to 5 month. Arterial lesions are quantifiedin serial cryosections of the proximal aorta by staining with Oil Red 0and counterstaining with hematoxylin. Lesion area size is determinedusing an digital imaging system.

EXAMPLE 14

In Vivo Testing of Cardiovascular Effects of Test Compounds

Hemodynamics in Anesthetized Rats

Male Wistar rats weighing 300-350 g (Harlan Winkelmann, Borchen,Germany) are anesthetized with thiopental “Nycomed” (Nycomed, Munich,Germany) 100 mg kg⁻¹ i.p. A tracheotomy is performed, and catheters areinserted into the femoral artery for blood pressure and heart ratemeasurements (Gould pressure transducer and recorder, model RS 3400) andinto the femoral vein for substance administration. The animals areventilated with room air and their body temperature is controlled. Testcompounds are administered orally or intravenously.

Hemodynamics in Conscious SHR

Female conscious SHR (Moellegaard/Denmark, 220-290 g) are equipped withimplantable radiotelemetry, and a data aquisition system (Data Sciences,St. Paul, Minn., USA), comprising a chronically implantabletransducer/transmitter unit equipped with a fluid-filled catheter isused. The transmitter is implanted into the peritoneal cavity, and thesensing catheter is inserted into the descending aorta.

Single administration of test compounds is performed as a solution inTranscutol®/Cremophor®/H₂O (10/20/70=v/v/v) given orally by gavage. Theanimals of control groups only receive the vehicle. Before treatment,mean blood pressure and heart rate of treated and untreated controlgroups are measured.

Hemodynamics in Anesthetized Dogs

Studies are performed on anesthetized dogs of either sex (body weightbetween 20-30 kg). Anesthesia is initiated by slow intravenous injectionof 25 mg kg⁻¹ sodium thiopental (Trapanal®, Byk Gulden, Konstanz,Germany). The anesthesia is continued and maintained throughout theexperiment by continuous infusion of 0.04 mg kg⁻¹ h⁻¹ fentanyl(Fentanyl®, Janssen, Neuss, Germany) and 0.25 mg kg⁻¹ h⁻¹ droperidol(Dihydrobenzperidol, Janssen, Neuss, Germany). During this anaesthesia,heart rate is as low as 35-40 bpm due to increased vagal tone.Therefore, a parasympathetic blockade is achieved by intermittentinjections of atropine (0.1 mg per animal) (AtropinsulfatR, Eifelfango,Bad Neuenahr, Germany). After intubation the animals are artificiallyventilated at constant volume (EngströmR 300, Engström, Sweden) withroom air enriched with 30% oxygen to maintain an end-tidal CO₂concentration of about 5% (NormocapR, Datex, Finland).

The following catheters are implanted for measurement of cardiovascularparameters: a tip catheter for recording of left ventricular pressure isinserted into the ventricle via the carotid artery (PC350, MillarInstruments, Houston, Tex., USA), a hollow catheter is inserted into thefemoral artery and connected to a strain gauge (type 4-327-1, TelosMedical, Upland, Calif., USA for recording of arterial blood pressure,two venous catheters are inserted into either femoral vein and oneadditional catheter into a forearm vein for application of theanesthetic and drugs, respectively, and an oxymetry catheter forrecording of oxygen saturation is inserted into the coronary sinus viathe jugular vein (Schwarzer IVH4, München, Germany).

After a left-sided thoracotomy the ramus circumflexus of the leftcoronary artery (LCX) is freed from connective tissue, and anelectromagnetic flow probe (Gould Statham, Oxnard, Calif., USA) isapplied for measurement of coronary blood flow. Arterial blood pressure,electrocardiogram (lead II), left ventricular pressure, first derivativeof left ventricular pressure (dP/dt), heart rate, coronary blood flow,and oxygen saturation in the coronary sinus are continuously recorded ona pen recorder (Brush, Gould, Cleveland, Ohio, USA). The maximum ofdP/dt is used as measure of left ventricular contractility (dP/dtmax).After completion of the instrumentation, an interval of 60 min isallowed for stabilization before the test compound is intravenouslyapplied as bolus injections. Care is taken that all measuredcardiovascular parameters have returned to control level beforeinjection of the next dose. Each dose of the test compound is tested atleast three times in different animals. The order of injection of thedifferent doses is randomized in each animal.

EXAMPLE 15

In Vivo Testing of Compounds/Target Validation for the Treatment ofInflammatory Disorders

Mouse Anti-CD3 Induced Cytokine Production Model

BALB/c mice are injected with a single intravenous injection of 10 μg of145-2C11 (purified hamster anti-mouse CD3ε monoclonal antibodies,PHARMINGEN). A test compound is administered intraperitoneally 60 minprior to the anti-CD3 mAb injection. Blood is collected 90 minutes afterthe antibody injection. Serum is obtained by centrifugation at 3000 rpm.for 10 min. IL-2 and IL-4 levels in the serum are determined by anELISA.

Mouse Anti-IgD Induced IgE Production Model

BALB/c mice are injected intravenously with 0.8 mg of purified goatanti-mouse IgD antibody or PBS (defined as day 0). Compound isadministered intraperitoneally from day 0 to day 6. On day 7 blood iscollected and serum is obtained by centrifugation at 3000 rpm. for 10min. Serum total levels of IgE are determined by YAMASA's ELISA kit andtheir Ig subtypes are done by an Ig ELISA KIT (Rougier Biotech's,Montreal, Canada).

Mouse LPS-Induced TNF-α Production Model

BALB/c mice are injected intraperitoneally with LPS (200 μg/mouse).Compound is administered intraperitoneally 1 h before the LPS injection.Blood is collected at 90 min post-LPS injection and plasma is obtained.TNF-α concentration in the sample is determined using an ELISA it.

Mouse Eotaxin-Induced Eosinophilia Model

BALB/c mice are injected intradermally with a 2.5 ml of air on days −6and −3 to prepare airpouch. On day 0 compound is administeredintraperitoneally 60 min before eotaxin injection (3 μg/mouse, i.d.).IL-5 (300 ng/mouse) is injected intravenously 30 min before the eotaxininjection. After 4 h of the eotaxin injection leukocytes in exudate iscollected and the number of total cells is counted. The differentialcell counts in the exudate are performed by staining with May-GrunwaldGimsa solution.

Mouse D10 Cell Transfer Model

D10.G4.1 cells (1×10⁷ cells/mouse) containing 2 mg of conalbumin insaline is administered i.v. to AKR mice. After 6 h blood is collectedand serum is obtained by centrifugation at 3000 rpm. for 10 min. IL-4and IL-5 level in serum are determined by ELISA kits. Compound isadministered intraperitoneally at 4 and +1 h after these cellsinjection.

Passive Cutaneous Anaphylaxis (PCA) Test in Rats

6 Weeks old male Wistar rats are sensitized intradermally (i.d.) ontheir shaved backs with 50 μl of 0.1 μg/ml mouse anti-DNP IgE monoclonalantibody (SPE-7) under a light anesthesia. After 24 hours, the rats arechallenged intravenously with 1 ml of saline containing 0.6 mg DNP-BSA(30) (LSL CO., LTD) and 0.005 g of Evans blue. Compounds are injectedintraperitoneally (i.p.) 0.5 h prior to antigen injection. Rats withoutthe sensitization, challenge, and compound treatment are used for ablank (control) and rats with sensitization, challenge and vehicletreatment are used to determine a value without inhibition. Thirty minafter the challenge, the rats are killed, and the skin of the back isremoved. Evans blue dye in the skin is extracted in formamide overnightat 63° C. Then an absorbance at 620 nm is measured to obtain the opticaldensity of the leaked dye.

Percent inhibition of PCA with a compound is calculated as follows:% inhibition={(mean vehicle value−sample value)/(mean vehicle value−meancontrol value)}×100Anaphylactic Bronchoconstriction in Rats

6 Weeks old male Wistar rats are sensitized intravenously (i.v.) with 10μg mouse anti-DNP IgE, SPE-7, and 1 days later, the rats are challengedintravenously with 0.3 ml of saline containing 1.5 mg DNP-BSA (30) underanesthesia with urethane (1000 mg/kg, i.p.) and gallamine (50 mg/kg,i.v.). The trachea is cannulated for artificial respiration (2ml/stroke, 70 strokes/min). Pulmonary inflation pressure (PIP) isrecorded through a side-arm of cannula connected to pressure transducer.Change in PIP reflects change of both resistance and compliance of thelungs. To evaluate the drugs, each drug is given i.v. 5 min beforechallenge.

EXAMPLE 16

In Vitro Testing of Compounds/Target Validation for the Treatment ofHematological Disorders

Isolation of CD34⁺ Cells

Mononuclear cells from fresh blood (cord blood, peripheral blood, bonemarrow) were separated by Ficoll Paque® (1.077 density,Amersham-Pharmacia) density gradient centrifugation, and CD34⁺ cellswere purified by immunomagnetic separation system (MiniMACS, MiltenyiBiotec), according to the manufacture's instructions (Direct CD34Progenitor Cell Isolation Kit, Miltenyi Biotec). The percentage of CD34⁺cells were generally from 90-95%.

Erythropoiesis/Anemia

Erythroid CD34⁺ Liquid Culture

1-2×10⁴ CD34⁺ cells were plated in triplicate in 24-well plates with 1ml Iscoves modified Dulbecco medium (IMDM) (Invitrogen) containing 10%fetal bovine serum (FCS, Invitrogen), 1% Glutamine (Invitrogen)supplemented with SCF (25 ng/ml) (PeproTech), different concentration ofErythropoietin (0.01 U/ml-1 U/ml) (Erypo® FS 4000, Cilag) with orwithout compounds. Control cells were incubated with 0.1-0.2% DMSOinstead of compounds. The cultures were incubated at 37° C. in a fullyhumidified atmosphere with 5% CO₂. After 9 to 14 days cells wereharvested, counted and stained with phycoerythrin (PE)-conjugated mAbagainst Glycophorin A (Pharmingen) to analyze differentiation.

Erythroid Colony-Forming Assay

Five hundred CD34⁺ cells/ml were plated in triplicate 24-well plateswith 1% methylcellulose in IMDM containing 30% FCS, 1% bovine serumalbumin (BSA), 2 mM L-glutamine and 10-4 M 2-mercaptoethanol (MethocultH4230, Cell Systems®, IL-3 (10 ng/ml) (PeproTech) with differentconcentration of erythropoietin (0.01 U/ml-1 U/ml) with or withoutcompounds. The cultures were incubated at 37° C. in a fully humidifiedatmosphere with 5% CO₂. After 9 to 14 days erythroid burst forming units(BFU-E) were counted from each of the plates. Afterwards cells weredissolved from methylcellulose with 0.1% NaCl solution. Cells werecounted and stained with phycoerythrin (PE)-conjugated mAb againstGlycophorin A (Pharmingen) to analyze differentiation.

BFU-E Culture

1×10⁵ Cord Blood CD34⁺ cells/ml were cultured in IMDM containing 15%BIT-9500 (Cell Systems®), supplemented with IL-3 (10 ng/ml), IL-6 (10ng/ml) and SCF (25 ng/ml) (PeproTech) and incubated at 37° C. in a fullyhumidified atmosphere with 5% CO₂. 3 and 5 days after initiation ofculture an equal volume of fresh medium supplemented with 2× cytokineswere added. On day 6 to 7 1-2×10⁴ erytroid progenitors were plated intriplicate in 24-well plates with 1 ml IMDM containing 10% FCS, 1%glutamine supplemented with SCF (25 ng/ml), different concentration oferythropoietin (0.01 U/ml-1 U/ml) with or without compounds. Controlcells were incubated with 0.1-0.2% DMSO instead of compounds. Thecultures were incubated at 37° C. in a fully humidified atmosphere with5% CO₂. After 6 to 8 days cells were harvested and counted to analyzeproliferation.

CD36⁺ Cells

1×10⁵ Cord Blood CD34⁺ cells/ml were cultured in IMDM containing 15%BIT-9500 supplemented with IL-3 (10 ng/ml), IL-6 (10 ng/ml) and SCF (25ng/ml) and incubated at 37° C. in a fully humidified atmosphere with 5%CO₂. 3 and 5 days after initiation of culture an equal volume of freshmedium supplemented with 2× cytokines were added. On day 6 to 7 cellswere stained with PE-conjugated mAb against CD36 (Pharmingen) and CD36⁺cells were purified using anti-PE microbeads and Mini MACS system(Miltenyi Biotec) according to the manufacture's instructions. 1-2×10⁴CD36⁺ cells were plated in triplicate 24-well plates with 1 ml IMDMcontaining 10% FCS, 1% Glutamine supplemented with SCF (25 ng/ml),different concentration of Erythropoietin (0.01 U/ml-1 U/ml) with orwithout compounds. Control cells were incubated with 0.1-0.2% DMSOinstead of compounds. The cultures were incubated at 37° C. in a fullyhumidified atmosphere with 5% CO₂. After 6 to 8 days cells wereharvested and counted to analyze proliferation.

Myelopoiesis and Thrombocytopoiesis

Myeloid CD34⁺ Liquid Culture

5×10³ CD34⁺ cells isolated from peripheral blood, cord blood or frombone marrow were pre-incubated in quadruplicate in 24-well plates in 1ml medium (StemSpan) with 15% FCS, SCF (20 ng/ml) and GM-CSF (2,5 ng/ml)for 6 to 7 days at 37° C. and 5.5% CO₂. Then compounds (0.1.1 or 10 μMin DMSO) with or without G-CSF (0.25 ng/ml; Neupogen®) were added andincubated for another 6 to 7 days. The number of the early myelopoieticCD15⁺/CD11b⁻ cells and the number of the late myelopoietic CD15⁺/CD11b⁺cells were determined by cell count (proliferation) and FACS(fluorescent associated cell sorting) analysis (differentiation) at day13-14.

Megakaryoid CD34⁺ Liquid Culture

5×10³ CD34⁺ cells isolated from peripheral blood, cord blood or frombone marrow were incubated in quadruplicate 24-well plates in 1 mlserum-free medium with 2% BSA, SCF (20 ng/ml) and compounds (0.1, 1 or10 μM in DMSO) with or without TPO (0-10 ng/ml) for 12 to 13 days at 37°C. and 5% CO₂. The number of the megakaryoid CD41⁺ cells (scatterprofile) were determined by FACS analysis. Megakaryocytes will beexamined by microscope if necessary.

In Vivo Testing of Compounds/Target Validation

Erythropoiesis/Anemia

Compounds which have demonstrated effects on the drug target in vitrohave been administered to normal or anemic animals orally orparenterally. In most cases, mice were used for compound testing. Insome cases, other species, e.g. rats, hamsters or guinea pigs have beenused in addition. Usually, repeated dosage is required for detection ofchanges in peripheral blood parameters. During the dosage period and upto five days after the last administration blood samples were drawn foranalysis of red and white blood cell counts as well as platelet countsusing an automated blood analyzer. In addition, erythropoiesis wasassessed by manual hematocrit and reticulocyte count determination. Forspecific analysis of leukocyte differentiation fluorescent associatedcell sorting SACS) was used.

Myelopoiesis and Thrombocytopoiesis

Myelopoiesis

Immunocompetent Balb/c mice were treated with compounds at differentdoses (based on pharmacokinetic data) once/day or bid per-orally orparenterally for up to 4 days. The WBC (white blood cells count) and theneutrophil count were monitored by FACS (CD11b⁺; scatter properties).

Immunocompromised Balb/c were generated by intravenous treatment with5-FU (100 mg/kg ip). 24 hours later the mice were treated with the testcompound at different doses (based on pharmacokinetic data) once/day orbid per-orally or parenterally for up to 7 to 13 days. Peripheral bloodcounts (WBC, RBC, PLT) have been determined after retroorbital plexuspuncture at days 5, 7, 11 and 14. For more detailed investigations thedevelopment of cellularity of femural bone marrow and spleen wereinvestigated by FACS analysis. The expression of specificdifferentiation markers on stem and progenitor cells (e.g. CD34, CD33,CD38, CD11b) and scatter properties were investigated.

Thrombocytopoiesis

Thrombopoietic compounds at different doses (based on pharmacokineticdata) were administered orally or parenterally following chemotherapy(Carboplatin, 100 mg/kg ip) immunocompromised mice. After repeatedadministration (once/day or bid for five to seven days) peripheral bloodplatelets (automated blood analyzer) have been determined afterretroorbital plexus puncture at day 5, 7, 11, and 14.

EXAMPLE 17

Bladder Outlet Obstruction Model for Assessing the Treatment ofGenito-Urinary Diseases

Wistar rats (200˜250 g/Charles River Japan) are anesthetizedintraperitoneally with ketamine. The abdomen is opened through a midlineincision and the bladder and the proximal urethra are exposed. Aconstant degree of urethral obstruction is produced by tying a ligaturearound the urethra and a catheter with an outer diameter of 1 mm. Theabdominal well is closed and the animals allowed to recover.

After 6 weeks, the rats are anesthetized with ketamine, and the ligaturearound the urethra is carefully removed to normalize the outletresistance and enable repetitive micturition. A polyethylene catheter isimplanted in the bladder through the dome, and exteriorized at thescapular level. Animals are then allowed to recover for at least 48hours.

Cytometric investigation is performed without anesthesia two days afterbladder catheter implantation in control and obstructed animals. Thebladder catheter was connected via a T-tube to a strain gauge and amicroinjection pump. The conscious rats are held under partial restraintin a restraining device. Warmed saline is infused into the bladder at arate of 3 ml/h for control and obstructed animals. The rate of infusionis increased from 3 to 10 ml/h to obtain similar interval times betweenmicturitions in obstructed and control rats. Overactivity of theobstructed bladders is assessed by measuring the cystometric parameterssuch as basal pressure, peak micturition pressure, threshold pressure,micturition interval, amplitude and frequency of spontaneous activityand micturition slope. Lluel et al., J. Urol. 160, 2253-57, 1998.

A test compound is dissolved in an appropriate vehicle, such as amixture of ethanol, Tween 80 (ICN Biomedicals Inc.), and saline (1:1:8,v/v/v), is administered intravenously through the catheter.

Organ Bath Assay for Measuring Agonist-Induced Contraction of Prostatefor Assessing the Treatment of Genito-Urinary Diseases.

An organ bath assay is employed to measure the agonist-inducedcontraction of prostate for assessing the biological activity of testcompounds (i.e., drug candidates). Male Wistar rats (200˜250 g/CharlesRiver Japan) are anesthetized with ether and sacrificed by dislocatingthe necks. The whole prostate is excised and placed in oxygenatedModified Krebs-Henseleit solution (pH 7.4) of the following composition(112 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 2 mM CaCl₂, 2.5mM NaHCO₃, 12 mM glucose). Ventricle prostate lobes were dissected intoseveral strips depending on the size of prostate. Prostate strips areequilibrated for 60 min in organ bath chambers before any stimulation.

Isometric tension is recorded under an appropriate load. Contractileresponse to adrenergic agonists or electric field stimulation isdetermined several times until reproducible responses are obtained. Testcompounds are pre-incubated prior to the agonistic or electricstimulation. The ratio of each contraction to the negative control iscalculated and the effect of the test compounds on the prostatecontraction is evaluated.

Organ Bath Assay for Measuring Agonist-Induced Contraction of UrinaryBladder for Assessing the Treatment of Genito-Urinary Diseases

An organ bath assay is employed to measure the agonist-inducedcontraction of urinary bladder for assessing the biological activity oftest compounds (i.e., drug candidates). Male Wistar rats (200˜250g/Charles River Japan) are anesthetized with ether and sacrificed bydislocating the necks. The whole urinary bladder is excised and placedin oxygenated Modified Krebs-Henseleit solution (pH 7.4) of thefollowing composition (112 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.2 mMNaH₂PO₄, 2 mM CaCl₂, 2.5 mM NaHCO₃, 12 mM glucose).

Isometric tension is recorded under an appropriate load usinglongitudinal strips of rat detrusor muscle. Bladder strips areequilibrated for 60 minutes before each stimulation. Contractileresponse to 80 mM KCl is determined at 15 minute intervals untilreproducible responses are obtained. The response to KCl is used as aninternal standard to evaluate the effect of test compounds.

The effects of test compounds are investigated by incubating the stripswith compounds for 30 minutes prior to stimulation with an appropriateagonist or electrical stimulation. One of the preparations made from thesame animal serves as a control, while others are used for evaluatingtest compounds. The ratio of each contraction to the internal standard(e.g., a KCl-induced contraction) is calculated, and the effects of thetest compounds on the contraction are evaluated.

EXAMPLE 18

In Vivo Testing of Compounds/Target Validation for the Treatment ofGenito-Urinary Diseases

-   1. Animals. Female Sprague-Dawley rats (200˜250 g/Charles River    Japan) are used.-   2. Catheter implantation. Rats are anesthetized by intraperitoneal    administration of urethane (Sigma) at 1.25 g/kg. The abdomen is    opened through a midline incision, and a polyethylene catheter    (BECTON DICKINSON, PE50) is implanted into the bladder through the    dome. In parallel, the inguinal region is incised, and a    polyethylene catheter (BECTON DICKINSON, PE50) filled with saline    (Otsuka) is inserted into a femoral vein.-   3. Investigation of bladder contraction. The bladder is filled via    the catheter by incremental volume of saline until spontaneous    bladder contractions occur. The intravesicular pressure is measured    a pressure transducer and displayed continuously on a chart    recorder. The activity of test compounds is assessed after    intravenous administration through a polyethylene cannula inserted    into the femoral vein.    Measurement of Bladder Cystometry in Conscious Rats-   1. Animals. Female Sprague-Dawley rats (200˜250 g/Charles River    Japan) are used.-   2. Catheter implantation. Rats are anesthetized by intramuscular    administration of ketamine (75 mg/kg) and xylazine (15 mg/kg). The    abdomen is opened through a midline incision, and a polyethylene    catheter (BECTON DICKINSON, PE50) is implanted into the bladder    through the dome. The catheter is tunneled through subcutis of the    animal by needle (14G) to neck. In parallel, the inguinal region is    incised, and a polyethylene catheter (BECTON DICKINSON, PESO) filled    with saline (Otsuka) is inserted into a femoral vein. The catheter    is tunneled through subcutis of the animal by needle to neck.-   3. Cystometric investigation. The bladder catheter is connected via    T-tube to a pressure transducer (Viggo-Spectramed Pte Ltd, DT-XXAD)    and a microinjection pump (TERUMO). Saline is infused at room    temperature into the bladder at a rate of 10 ml/hr. Intravesicular    pressure is recorded continuously on a chart pen recorder    (Yokogawa). At least three reproducible micturition cycles are    recorded before a test compound administration.-   4. Administration of test compounds. A test compound dissolved in    the mixture of ethanol, Tween 80 (ICN Biomedicals Inc.) and saline    (1:1:8, v/v/v) is administered intravenously through the catheter.

EXAMPLE 20

Expression Profiling

Total cellular RNA was isolated from cells by one of two standardmethods: 1) guanidine isothiocyanate/cesium chloride density gradientcentrifugation [Kellogg et al. (1990)]; or with the Tri-Reagent protocolaccording to the manufacturer's specifications (Molecular ResearchCenter, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagentprotocol was treated with DNAse I to remove genomic DNA contamination.

For relative quantitation of the mRNA distribution, total RNA from eachcell or tissue source was first reverse transcribed. 85 μg of total RNAwas reverse transcribed using 1 μmole random hexamer primers, 0.5 mMeach of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany) and 3000 URnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680μl. The first strand synthesis buffer and Omniscript reversetranscriptase (2 U/μl) were obtained from (Qiagen, Hilden, Germany). Thereaction was incubated at 37° C. for 90 minutes and cooled on ice. Thevolume was adjusted to 6800 μl with water, yielding a finalconcentration of 12.5 ng/μl of starting RNA.

For relative quantitation of the distribution of mRNA in cells andtissues the Perkin Elmer ABI Prism RTM 7700 Sequence Detection system orBiorad iCycler was used according to the manufacturer's specificationsand protocols. PCR reactions were set up to quantitate expression of thetest gene and the housekeeping genes HPRT (hypoxanthinephosphoribosyltransferase), GAPDH (glyceraldehyde-3-phosphatedehydrogenase), β-actin, and others. Forward and reverse primers andprobes were designed using the Perkin Elmer ABI Primer Express™ softwareand were synthesized by TibMolBiol (Berlin, Germany). The forward primersequence was: Primerl cgtgttaacgaacagcctga (SEQ ID NO:19). The reverseprimer sequence was Primer2 caactgaagaccgccatctc (SEQ ID NO:20). Probelccgtgctaactgttccaggcctgac (SEQ ID NO:21), labeled with FAM(carboxyfluorescein succinimidyl ester) as the reporter dye and TAMRA(carboxytetramethylrhodamine) as the quencher, was used as a probe. Thefollowing reagents were prepared in a total of 25 μl: 1× TaqMan bufferA, 5.5 mM MgCl₂, 200 nM of DATP, dCTP, dGTP, and dUTP, 0.025 U/μlAmpliTaq Gold™, 0.01 U/μl AmpErase, and Probelccgtgctaactgttccaggcctgac, forward and reverse primers each at 200 nM,200 nM, FAM/TAMRA-labeled probe, and 5 μl of template cDNA. Thermalcycling parameters were 2 min at 50° C., followed by 10 min at 95° C.,followed by 40 cycles of melting at 95° C. for 15 sec andannealing/extending at 60° C. for 1 min.

Calculation of Corrected CT Values

The CT (threshold cycle) value is calculated as described in the“Quantitative determination of nucleic acids” section. The CF-value(factor for threshold cycle correction) is calculated as follows:

-   1. PCR reactions were set up to quantitate the housekeeping genes    (HKG) for each cDNA sample.-   2. CT_(HKG)-values (threshold cycle for housekeeping gene) were    calculated as described in the “Quantitative determination of    nucleic acids” section.-   3. CT_(HKG)-mean values (CT mean value of all HKG tested on one    cDNAs) of all HKG for each cDNA are calculated (n=number of HKG):    CT _(HKG-n)-mean value ═(CT _(HKG1)-value+CT _(HKG2)-value+ . . .    +CT _(HKG-n)-value)/n    -   4. CT_(pannel) mean value (CT mean value of all HKG in all        tested cDNAs)=(CT_(HKG1)-mean value+CT_(HKG2)-mean value+ . . .        +CT_(HKG-y)-mean value)/y (y=number of cDNAs)-   5. CF_(cDNA-n) (correction factor for cDNA n)=CT_(pannel)-mean value    —CT_(HKG-n)-mean value-   6. CT_(cDNA-n) (CT value of the tested gene for the cDNA    n)+CF_(cDNA-n) (correction factor for cDNA n)=CT _(cor-cDNA-n)    (corrected CT value for a gene on cDNA n)    Calculation of Relative Expression    Definition: highest CT_(cor-cDNA-n)≠40 is defined as CT_(cor-cDNA)    [high]    Relative Expression=2^((CTcor-cDNA[high]−CTcor-cDNA-n))

Expression was tested in the following tissues: fetal heart, heart,pericardium, heart atrium (right), heart atrium (left), heart ventricle(left), heart ventricle (right), heart apex, Purkinje fibers,interventricular septum, fetal aorta, aorta, aorta sclerotic, artery,coronary artery, coronary artery sclerotic, pulmonary artery, carotidartery, mesenteric artery, vein, pulmonic valve, coronary artery smoothmuscle primary cells, HUVEC cells, skin, adrenal gland, thyroid, thyroidtumor, pancreas, pancreas liver cirrhosis, esophagus, esophagus tumor,stomach, stomach tumor, colon, colon tumor, small intestine, ileum,ileum tumor, ileum chronic inflammation, rectum, salivary gland, fetalliver, liver, liver cirrhosis, liver tumor, HEP G2 cells, leukocytes(peripheral blood), Jurkat (T-cells), bone marrow, erythrocytes, lymphnode, thymus, thrombocytes, bone marrow stromal cells, bone marrow CD71⁺cells, bone marrow CD33⁺ cells, bone marrow CD34⁺ cells, bone marrowCD15⁺ cells, cord blood CD71⁺ cells, cord blood CD34⁺ cells, neutrophilscord blood, neutrophils peripheral blood, spleen, spleen livercirrhosis, skeletal muscle, adipose, fetal brain, brain, Alzheimerbrain, cerebellum, cerebellum (right), cerebellum (left), cerebralcortex, Alzheimer cerebral cortex, frontal lobe, Alzheimer brain frontallobe, occipital lobe, parietal lobe, temporal lobe, precentral gyrus,postcentral gyrus, tonsilla cerebefli, vermis cerebelli, pons,substantia nigra, cerebral meninges, cerebral peduncles, corpuscallosum, hippocampus, thalamus, dorsal root ganglia, spinal cord,neuroblastoma SK-N-MC cells, neuroblastoma SH-SY5Y cells, neuroblastomaIMR32 cells, glial tumor H4 cells, glial tumor H4 cells+APP, HEK CNS,HBEK CNS+APP, retina, fetal lung, fetal lung fibroblast IR-90 cells,fetal lung fibroblast MRC-5 cells, lung, lung right upper lobe, lungright mid lobe, lung right lower lobe, lung lupus disease, lung tumor,lung COPD, trachea, cervix, testis, HeLa cells (cervix tumor), placenta,uterus, uterus tumor, ovary, ovary tumor, breast, breast tumor, MDA MB231 cells (breast tumor), mammary gland, prostate, prostate BPH,bladder, ureter, penis, corpus cavemosum, fetal kidney, kidney, kidneytumor, and HEK 293 cells.

Expression Profile

The results of the mRNA-quantification (expression profiling) is shownin Table 1. TABLE 1 Tissue Relative Expression fetal heart 474 heart 512pericardium 443 heart atrium (right) 1370 heart atrium (left) 904 heartventricle (left) 160 heart ventricle (right) 30 heart apex 4124 Purkinjefibers 1552 interventricular septum 744 fetal aorta 11 aorta 51 aortasclerotic 20 artery 25 coronary artery 237 coronary artery sclerotic 61pulmonary artery 69 carotid artery 55 mesenteric artery 66 vein 16pulmonic valve 184 coronary artery smooth muscle primary cells 1 HUVECcells 867 skin 873 adrenal gland 4738 thyroid 613 thyroid tumor 304pancreas 189 pancreas liver cirrhosis 676 esophagus 16 esophagus tumor1389 stomach 955 stomach tumor 2837 colon 461 colon tumor 103 smallintestine 1458 ileum 891 ileum tumor 131 ileum chronic inflammation 4rectum 1105 salivary gland 193 fetal liver 572 liver 949 liver cirrhosis724 liver tumor 3902 HEP G2 cells 9345 leukocytes (peripheral blood) 218Jurkat (T-cells) 60 bone marrow 179 erythrocytes 18 lymph node 62 thymus413 thrombocytes 16 bone marrow stromal cells 3304 bone marrow CD71⁺cells 4 bone marrow CD33⁺ cells 21 bone marrow CD34⁺ cells 93 bonemarrow CD15⁺ cells 0 cord blood CD71⁺ cells 0 cord blood CD34⁺ cells 416neutrophils cord blood 402 neutrophils peripheral blood 0 spleen 1113spleen liver cirrhosis 1038 skeletal muscle 197 adipose 191 fetal brain505 brain 917 Alzheimer brain 1479 cerebellum 76 cerebellum (right) 1160cerebellum (left) 1820 cerebral cortex 2521 Alzheimer cerebral cortex3019 frontal lobe 3169 Alzheimer brain frontal lobe 3591 occipital lobe1965 parietal lobe 1663 temporal lobe 2419 precentral gyrus 1184postcentral gyrus 16 tonsilla cerebelli 410 vermis cerebelli 385 pons1687 substantia nigra 7082 cerebral meninges 17 cerebral peduncles 252corpus callosum 1531 hippocampus 2452 thalamus 657 dorsal root ganglia43 spinal cord 1269 neuroblastoma SK-N-MC cells 28 neuroblastoma SH-SY5Ycells 1209 neuroblastoma IMR32 cells 30 glial tumor H4 cells 90 glialtumor H4 cells + APP 191 HEK CNS 910 HEK CNS + APP 879 retina 116 fetallung 1252 fetal lung fibroblast IMR-90 cells 10 fetal lung fibroblastMRC-5 cells 3 lung 347 lung right upper lobe 617 lung right mid lobe 326lung right lower lobe 592 lung lupus disease 313 lung tumor 223 lungCOPD 377 trachea 734 cervix 309 testis 1305 HeLa cells (cervix tumor) 3placenta 976 uterus 491 uterus tumor 1924 ovary 2034 ovary tumor 2504breast 2487 breast tumor 343 MDA MB 231 cells (breast tumor) 413 mammarygland 1520 prostate 1389 prostate BPH 217 bladder 560 ureter 495 penis51 corpus cavernosum 247 fetal kidney 2385 kidney 1209 kidney tumor14869 HEK 293 cells 781

REFERENCES

-   Graham D K, Dawson T L, Mullaney D L, Snodgrass H R, Earp H S.    Cloning and mRNA expression analysis of a novel human protooncogene,    c-mer. Cell Growth Differ 1994 June; 5-   Evans C O, Young A N, Brown M R, Brat D J, Parks J S, Neish A S,    Oyesiku N M. Novel patterns of gene expression in pituitary adenomas    identified by complementary deoxyribonucleic acid microarrays and    quantitative reverse transcription-polymerase chain reaction. J Clin    Endocrinol Metab 2001 July;86(7):3097-107-   Georgescu M M, Kirsch K H, Shishido T, Zong C, Hanafusa H.    Biological effects of c-Mer receptor tyrosine kinase MerTK in    hematopoietic cells depend on the Grb2 binding site in the receptor    and activation of NF-kappaB. Mol Cell Biol 1999 February;    19(2):1171-81

1-17. (canceled)
 18. An isolated and purified protein comprising theamino acid sequence shown in SEQ ID NO:2.
 19. An isolated and purifiedprotein comprising an amino acid sequence which is at least 95%identical to the amino acid sequence of claim 18 and which has areceptor tyrosine kinase MerTK activity.
 20. A purified preparation ofantibodies which specifically bind to the protein of claim
 18. 21. Thepreparation of claim 20 wherein the antibodies are polyclonal.
 22. Thepreparation of claim 20 wherein the antibodies are monoclonal.
 23. Thepreparation of claim 20 wherein the antibodies are single-chainantibodies.
 24. The preparation of claim 20 wherein the antibodies areFab, F(ab′)₂, or Fv fragments.
 25. An isolated and purifiedpolynucleotide which encodes the protein of claim
 18. 26. Thepolynucleotide of claim 25 which comprises the nucleotide sequence shownin SEQ ID NO:1 or SEQ ID NO:3.
 27. The polynucleotide of claim 25 whichis a cDNA.
 28. An isolated and purified single-stranded polynucleotidecomprising at least 8 contiguous nucleotides of a coding sequence or acomplement of the coding sequence for the protein of claim
 18. 29. Thepolynucleotide of claim 28 wherein the coding sequence comprises thenucleotide sequence shown in SEQ ID NO:1 or
 3. 30. An expressionconstruct, comprising; a coding sequence for the protein of claim 18;and a promoter which is located upstream from the coding sequence andwhich controls expression of the coding sequence.
 31. The expressionconstruct of claim 30 wherein the coding sequence comprises thenucleotide sequence of SEQ ID NO:1 or
 3. 32. A host cell comprising theexpression construct of claim
 30. 33. The host cell of claim 32 which isprokaryotic.
 34. The host cell of claim 32 which is eukaryotic.
 35. Amethod of producing a protein, comprising the steps of: culturing thehost cell of claim 32 under conditions whereby the protein is expressed;and recovering the protein.
 36. A method of detecting an expressionproduct of a gene encoding the protein of claim 18, comprising the stepsof: contacting a test sample with a reagent that specifically binds toan expression product of the nucleotide sequence shown in SEQ ID NO: 1or 3; assaying the test sample to detect binding between the reagent andthe expression product; and identifying the test sample as containingthe expression product if binding between the reagent and the expressionproduct is detected.
 37. The method of claim 36 wherein the expressionproduct is a protein.
 38. The method of claim 36 wherein the reagent isan antibody.
 39. The method of claim 36 wherein the cell is cultured invitro and wherein the test sample is culture medium.
 40. The method ofclaim 36 wherein the expression product is an mRNA molecule.
 41. Themethod of claim 40 wherein the reagent is an antisense oligonucleotide.42. A method of treating comprising the step of: administering to apatient having a disorder selected from the group consisting of agastrointestinal disorder, a liver disorder, a metabolic disorder, aneurological disorder, a cardiovascular disorder, a hematologicaldisorder, a reproductive disorder, and endocrine disorder, a hormonaldisorder, a respiratory disorder, and a genitourinary disorder aneffective amount of an agent selected from the group consisting of anantisense oligonucleotide that regulates expression of a gene encodingthe protein of claim 18 and an antibody which specifically binds to theprotein of claim
 18. 43. A method of screening for candidate therapeuticagents, comprising the steps of: contacting the protein of claim 18 witha test compound; assaying for binding between the protein and the testcompound; and identifying a test compound that binds to the protein as acandidate therapeutic agent that may be useful for treating a disorderselected from the group consisting of a gastrointestinal disorder, aliver disorder, a metabolic disorder, a neurological disorder, acardiovascular disorder, a hematological disorder, a reproductivedisorder, and endocrine disorder, a hormonal disorder, a respiratorydisorder, and a genitourinary disorder.
 44. The method of claim 43wherein either the test compound or the protein comprises a detectablelabel.
 45. The method of claim 43 wherein either the test compound orthe protein is bound to a solid support.
 46. A method of screening forcandidate therapeutic agents, comprising the steps of: assaying forexpression of a polynucleotide encoding the protein of claim 18 in thepresence and absence of a test compound; and identifying a test compoundthat regulates the expression as a candidate therapeutic agent that maybe useful for treating a disorder selected from the group consisting ofa gastrointestinal disorder, a liver disorder, a metabolic disorder, aneurological disorder, a cardiovascular disorder, a hematologicaldisorder, a reproductive disorder, and endocrine disorder, a hormonaldisorder, a respiratory disorder, and a genitourinary disorder.
 47. Themethod of claim 46 wherein the step of contacting is in a cell.
 48. Themethod of claim 46 wherein the step of contacting is in a cell-free invitro translation system.
 49. A pharmaceutical composition comprising atherapeutic reagent selected from the group consisting of thepreparation of claim 20, an antisense oligonucleotide which binds to anexpression product of a gene which encodes the protein of claim 18, theprotein of claim 18, and the polynucleotide of claim 25; and apharmaceutically acceptable carrier.
 50. The pharmaceutical compositionof claim 49 wherein the polynucleotide comprises the nucleotide sequenceshown in SEQ ID NO:1 or 3.